Methods for preserving nucleated mammalian cells

ABSTRACT

Methods and compositions are provided for increasing the survival of nucleated mammalian cells following drying and rehydration. The methods include introducing a disaccharide such as trehalose into said cells, optionally including heat shock proteins, apoptosis inhibitors, and arbutin, drying said cells, and rehydrating them. The invention further provides nucleated mammalian cells that have increased capacity to survive, divide and, in some cases, differentiate, following drying and rehydration. The cells comprise a disaccharide and one or more of the following: a heat shock protein, an apoptosis inhibitor, and arbutin.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Federal support under Grant Nos.N66001-00-C-8048 and N66001-02-C-8055 awarded by the Defense AdvancedResearch Projects Agency and Grant No. HL57810 and HL61204 awarded bythe National Institutes of Health. The Government has certain rights inthe invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This patent application claims priority from U.S. patent applicationSer. No. 10/686,904, filed Oct. 16, 2003, U.S. patent application Ser.No. 10/721,557, filed Nov. 25, 2003, U.S. patent application Ser. No.10/721,678, filed Nov. 25, 2003 and U.S. patent application Ser. No.10/722,154, filed Nov. 25, 2003. The contents of these applications arehereby incorporated by reference.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK.

NOT APPLICABLE

FIELD OF THE INVENTION

Embodiments of the present invention generally broadly relate tobiological samples, such as mammalian (e.g., human) nucleated cells,such as stem cells, epithelial cells, and cells of the immune system.More specifically, embodiments of the present invention generallyprovide for the preservation and survival of such cells.

Embodiments of the present invention also generally broadly relate tothe therapeutic and in vitro uses of biological samples; moreparticularly to manipulations or modifications of biological samples,-such as loading biological samples with solutes (e.g., carbohydrates,such as trehalose) and preparing dried compositions that can bere-hydrated at the time of application. When dried biological samples ofthe present invention are rehydrated, they are restored to viability.

BACKGROUND OF THE INVENTION

Transporting and storing mammalian cells for in vitro and in vivo usehas been difficult due to the need of the cells for acceptabletemperatures, continued nutrients, and in some cases, reduced oxygentension. Currently, nucleated mammalian cells are stored by freezingthem in liquid nitrogen vapor, which requires introduction of acryoprotectant, such as dimethyl sulfoxide (DMSO) into the cells, andfreezing them to approximately −152° C. Besides the bulky equipment andsupplies needed for such storage, this process creates other problems.At the concentrations required to serve as a cyroprotectant, DMSO istoxic to cells at physiological temperatures due to hydrophobicinteractions with the proteins and membranes, and thus extensive washingof the cells is required following thawing. The thawing and washingprocedures can reduce cellular viability and recovery, which could thenaffect clinical efficacy.

Dehydrating cells represents an alternative to current approaches tostoring cells. It has been shown effective for the storage of humanblood platelets at room temperature for up to 2 years, during which timerecovery and response to thrombin remained essentially unchanged.Unfortunately, methods that are useful for platelets and methods thatare useful for red blood cells are not useful for nucleated mammaliancells. Efforts to dry nucleated cells have also been reported, butachieving consistent results of highly viable, physiologically activecells following dehydration to low water contents remains elusive.

The dehydration and rehydration steps themselves are extremely stressfulto the biological samples, thus protective compounds are required tosafeguard the membranes and proteins during these procedures, analogousto the use of cryoprotectants during freeze-thaw cycles. Trehalose, adisaccharide found in high concentrations in many desiccation-tolerantanimals and plants has been the excipient of choice for many cellulardehydration studies, due to its ability to replace the hydrogen bondedwater molecules in the dehydrated samples, its high glass transitiontemperature, and the stability of the glycosidic bond. Unfortunately,mammalian cells lack a transporter for trehalose, and various methods,such as inducing pores in the cells for brief periods or transfectingcells, have been tried in attempts to load mammalian cells withtrehalose in amounts sufficient to provide protection during drying andrehydration.

It would be desirable to have improved methods for dehydrating andrehydrating nucleated cells. The present invention fills these and otherneeds.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods and compositions for improving theviability and activity of mammalian nucleated cells that are dried andrehydrated.

In a first group of embodiments, the invention provides methods forloading a disaccharide into mammalian nucleated cells, comprising:contacting said cells for at least 2 hours with a solution comprising adisaccharide, thereby loading the cells with disaccharide to producedisaccharide-loaded mammalian nucleated cells. In some embodiments, thecells are stem cells, immune system cells, or epithelial cells. Thecontacting can be for 10 hours, or 24 hours. The disaccharide can be,for example, sucrose, maltose or trehalose, but is preferably trehalose.The solution can further comprise not more than 3% dimethyl sulfoxide.

In another group of embodiments, the invention provides methods forincreasing survival of mammalian nucleated cells following drying andrehydration, comprising: (a) contacting the cells with a solutioncomprising a disaccharide for at least 2 hours, thereby producingdisaccharide-loaded cells, (b) drying the disaccharide-loaded cells to aresidual water content between 0.2 and 0.5 gram water per gram of dryweight, and (c) rehydrating the cells, thereby increasing survival ofthe cells. The contacting may be for 24 hours. The cells may be, forexample, stem cells, immune system cells, or epithelial cells. Thedisaccharide can be, for example, sucrose, maltose or trehalose, but ispreferably trehalose. The cells may further comprise a heat shockprotein. The heat shock protein may be induced by exposing said cells toa heat shock. The heat shock may consist of raising the temperature ofmedium contacting the cells to 42-44° C. for one hour, and then allowingthe temperature of the medium to drop to 36-38° C. Alternatively, theheat shock protein may be introduced into the cells by contacting thecells with a solution comprising the protein. Further, the heat shockprotein may be expressed from a nucleic acid sequence introduced intothe cells. The heat shock protein may be p26 from Artemia franciscana.The cells may be contacted with a solution comprising an apoptosisinhibitor. The apoptosis inhibitor may be selected from the groupconsisting of N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methylketone (in which the aspartyl residue is o-methylated ornon-o-methylated), caspase I inhibitor II, calpain inhibitor, andBcl-xL. Further, the cells may be contacted by a solution comprisingarbutin or hydroquinone, provided that said cells are not 293 cells or Bcells. The cells may also be contacted by a solution comprising not morethan 3% dimethyl sulfoxide. In some embodiments, the cells are contactedby a solution comprising both a heat shock protein and an apoptosisinhibitor. The solution may further comprise not more than 3% dimethylsulfoxide. Cells contacted with a solution comprising arbutin orhydroquinone are preferably dried in a medium comprising arbutin orhydroquinone. The cells are preferably dried in rounded droplets ofdrying buffer.

In yet a further set of embodiments, the invention provides methods forincreasing survival of mammalian nucleated cells following drying andrehydration, comprising: (a) contacting the cells with a solutioncomprising an apoptosis inhibitor, thereby loading the cells with theapoptosis inhibitor, to produce apoptosis inhibitor-loaded cells, (b)drying said apoptosis inhibitor-loaded cells, and (c) rehydrating thecells, thereby increasing survival of the cells. The apoptosis inhibitormay be, for example,N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone (inwhich the aspartyl residue is o-methylated or non-o-methylated), CaspaseI inhibitor II, Calpain inhibitor, and Bcl-xL. The cells may be, forexample, stem cells, immune system cells, and epithelial cells The cellsare preferably dried in droplets of drying buffer.

In yet a further set of embodiments, the invention provides methods forincreasing survival of mammalian nucleated cells following drying andrehydration, comprising: (a) introducing a heat shock protein into, orinducing production of a heat shock protein in, said cells, to produceheat shock protein-loaded cells, (b) drying said heat shockprotein-loaded cells, and (c) rehydrating the cells, therebly increasingsurvival of the cells. The heat shock protein may be p26 from Artemiafranciscana. The heat shock protein may be introduced into the cells byincubating the cells in a medium comprising the heat shock protein. Theheat shock protein may be induced in said cells by raising thetemperature of medium contacting the cells to 42-44° C. for one hour,and then allowing the temperature of the medium to lower to 36-38° C.The heat shock protein may be introduced into the cells by introducinginto the cells a nucleic acid sequence comprising a promoter operablylinked to a sequence encoding the heat shock protein. The cells can be,for example, stem cells, immune system cells, or epithelial cells. Thecells are preferably dried in droplets of drying buffer.

In yet a further set of embodiments, the invention provides methods forincreasing survival of mammalian nucleated cells following drying andrehydration, provided said cells are not 293 cells or B cells,comprising: (a) incubating said cells with a compound selected fromarbutin and hydroquinone, to produce arbutin- or hydroquinone-loadedcells, (b) drying the arbutin- or hydroquinone-loaded cells, and (c)rehydrating said cells, thereby increasing survival of the cells. Insome embodiments, the compound of step (a) is arbutin.

In yet a further set of embodiments, the invention provides isolatedmammalian nucleated cells comprising a disaccharide and a compoundselected from the group consisting of arbutin and hydroquinone. In someembodiments, the compound is arbutin. In some embodiments, the cell isdried. In some embodiments, the cell further comprises an apoptosisinhibitor. In some embodiments, the cell further comprises a heat shockprotein. The disaccharide can be, for example, sucrose, maltose ortrehalose or a mixture of these, but is preferably trehalose.

In yet a further set of embodiments, the invention provides isolateddried mammalian nucleated cells comprising a disaccharide and anexogenous heat shock protein. The disaccharide can be, for example,sucrose, maltose or trehalose or a mixture of these, but is preferablytrehalose.

In yet a further set of embodiments, the invention provides isolateddried mammalian nucleated cells comprising a disaccharide and anexogenous apoptosis inhibitor. The disaccharide can be, for example,sucrose, maltose or trehalose, but is preferably trehalose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of viability (%) subsequent to drying vs. watercontent (gm. water/gm. dry weight) after drying for transfected 293cells (T-293 cells) and for control 293 cells (293-cells), with both thetransfected 293 cells and the control 293 cells having no trehaloseinternally and with the drying buffer for both transfected 293 cells andcontrol 293 cells having no trehalose.

FIG. 2 is a graph of viability (%) subsequent to drying vs. watercontent (gm. water/gm. dry weight) after drying for transfected 293cells (T-293 cells) and for control 293 cells (293-cells), with thetransfected 293 cells and the control 293 cells both having no trehaloseinternally and with the drying buffer for both transfected 293 cells andcontrol 293 cells having 150 mM trehalose.

FIG. 3 is a graph of viability (%) subsequent to drying vs. watercontent (gm. water/gm. dry weight) after drying for transfected 293cells (T293 cells) and for control 293 cells (293-cells), with both thetransfected 293 cells and control 293 cells having trehalose internallyand with the drying buffer for both the transfected 293 cells andcontrol 293 cells having 150 mM trehalose.

FIG. 4 is a graph of the number of colonies formed after rehydration vswater content after drying the transfected 293 cells (T293 cells) andthe control 293 cells (293-cells) to 0.3 gm. water/gm. dry weight and to0.2 gm. water/gm. dry weight, with both the transfected 293 cells andthe control 293 cells having trehalose internally and with both havingabout 150 mM trehalose in the drying buffer.

FIG. 5 is a graph of survival (% viability) vs. water content (gm.water/gm. dry weight) for a first batch of p26-transfected 293 cells(T293 cells) after air drying and rehydration, and for a second batch ofp26-transfected 293 cells (T293 cells) after vacuum drying andrehydration, with both batches of the transfected 293 cells havingtrehalose internally and with the drying buffer for both batchescontaining 150 mM trehalose. Both batches were loaded with trehalose for24 hours by incubation at 37 C with 100 mM trehalose. The cells weredried by either air-drying or by vacuum drying and the viability afterrehydration was determined by trypan blue exclusion. Air drying wasconducted by at room temperature in a modified desiccator flushed withdry air at approximately 200 mL/min. Vacuum dried samples were placed ina vacuum chamber at room temperature and subjected to a vacuum ofapproximately 3 inches Hg. The vacuum dried samples show a significantlyleft-shifted curve compared to the air-dried samples, indicating muchhigher viability at lower water levels.

FIG. 6 is a graph of survival (% viability) vs. water content (gm.water/gm. dry weight) for a first batch of transfected 293 cells (T293cells) after air drying while in a thin film configuration and afterrehydration, and for a second batch of transfected 293 cells (T-293cells) after air drying while in a plurality of droplets configurationand after rehydration, with both batches of the transfected 293 cellshaving trehalose internally and with the drying buffer for both batchescontaining 150 mM trehalose.

FIG. 7 is a graph of survival (% viability) vs. water content (gm.water/gm. dry weight) for a first batch of transfected 293 cells (T293cells) after vacuum drying while in a thin film configuration and afterrehydration, and for a second batch of transfected 293 cells (293 cells)after vacuum drying while in a plurality of droplets configuration andafter rehydration, with both batches of the transfected 293 cells havingtrehalose internally and with the drying buffer for both batchescontaining 150 mM trehalose.

FIG. 8 is a graph of survival (% average viability) vs. water content(gm. water/gm. dry weight) for 293 cells (293 cells) when vacuum driedin 50 μL droplets and after rehydration, and for transfected 293 cells(T293 cells) when vacuum dried in 50 μL and after rehydration, with boththe 293 cells and the transfected 293 cells having trehalose internallyand with the drying buffer for both batches containing 150 mM trehalose.Although both types show improved viability with this combinationcompared to air-dried samples, the transfected cells showed highersurvival than standard cells at water contents below 2 g H₂O/g dryweight.

FIGS. 9A and 9B are a flow chart showing preferred embodiments forperforming the methods of the invention using human cells.

DETAILED DESCRIPTION

I. Introduction

As noted in the Background, human nucleated cells lack a transporter fortrehalose, and a number of approaches have been tried to load such cellswith amounts of trehalose that will protect the cells during drying andsubsequent rehydration. Surprisingly, we have now discovered that cellscan be loaded with protective amounts of trehalose by endocytosis,provided that they incubated in a medium containing trehalose for asufficient time. We have further found that addition of certain otheragents to the medium (or, in the case of one group of agents, theinduction of the agents or transfection of the cells with the agents)result in yet further improvements in the percentage of the cells thatare viable after rehydration or in their ability to divide and, whereappropriate, differentiate. If desired, one or more of the other agentscan be combined in the cells to increase their survival following dryingand rehydration.

The methods provided herein are generally applicable to nucleatedmammalian cells, such as canine, feline, bovine, and equine cells, morepreferably primate cells, and even more preferably, human nucleatedcells. The methods of the invention provide the ability to dry suchcells to permit them to be transported and stored, and later rehydrated.The methods of the invention can be used to dry widely differing celltypes, such as (a) stem cells, including mesenchymal stem cells(“MSCs”), embryonic stem cells (“ESCs”) and cord blood stem cells(“CBSCs”), and cells that are partially differentiated from these cells,but which still retain the ability to further differentiate intoterminally differentiated cells, (b) immune system cells, such as Bcells, and (c) epithelial cells. Following drying by the methods of theinvention, the cells can be rehydrated and restored to viability.

Because living cells must generally be maintained under physiologicallyacceptable conditions (for example, conditions of temperature, ambientgas content and relative humidity suitable for tissue culture), cellsmust generally be transported quickly. This requires the use of courierservices and makes transport of cells over extended distances expensiveand logistically complex. The alternative to date has been to freeze thecells in liquid nitrogen. Cells frozen in liquid nitrogen must beshipped in containers which can safely hold liquid nitrogen or dry ice,which are bulky and which creates logistical difficulties. The abilityto dry mammalian nucleated cells for even a few days, to be able to shipthem under simple refrigeration, and to restore them to viabilitytherefore reduces the cost and difficulty of distributing the cells.

II. Methods of the Invention

A. Disaccharide Loading

Trehalose is known to stabilize cell membranes and proteins duringdehydration. The cells of humans and other mammals, however, do not havea transporter for trehalose. Accordingly, efforts to load trehalose intomammalian cells have typically employed methods to overcome thisproblem, such as by creating pores in the cells to allow entry oftrehalose or transfecting cells in an attempt to have them produce theirown trehalose. Such methods have typically involved brief exposure ofthe cells to the trehalose-containing medium, typically for ten tofifteen minutes and usually for less than an hour.

In one embodiment, the present invention relates to the surprisingdiscovery that mammalian cells incubated in a trehalose-containingmedium will take up trehalose by endocytosis. While simple, thisdiscovery has allowed us to load cells with trehalose without the morecomplicated procedures, such as creating pores in the cells, that theart has taught are necessary to get sufficient levels of intracellulartrehalose (e.g., from 15-50 mM trehalose) to be useful in drying thecells.

The methods are therefore suitable for any nucleated mammalian cell thathas sufficient endocytotic processes to take in enough trehalose from anextracellular medium during a 24 hour period to increase cell viabilitycompared to not having trehalose present. Whether any particularnucleated mammalian cell has sufficient endocytotic processes to take inadequate amounts of trehalose from an extracellular medium can bedetermined by routine assays, such as the lucifer yellow assay describedherein. Use of the term “biological samples” below refers to nucleatedmammalian cells. Similarly, the word “cells” as used herein, unlessotherwise indicated, refers to nucleated mammalian cells.

First, the cells of interest are incubated in a standard growth mediumcontaining trehalose to load the cells with trehalose (this medium willhereafter be referred to as the “incubation medium” or the “loadingbuffer”). Optionally, prior to the incubation, the cells may be culturedin a standard growth medium to increase the cell population. We havefound that incubation should be at least 10 hours, to give the cellsenough time to take up enough trehalose to be protective, but not muchover 48 hours, as the amount of trehalose taken into the cell plateaus,and cell viability begins to drop after 48 hours of incubation, as canbe determined by using a standard trypan blue viability assay.Therefore, the incubation is preferably from 10 to 48 hours, morepreferably from 15 to 40 hours, and yet more preferably from about 20hours to about 30 hours. An incubation of about 24 hours is preferred.

If desired, the incubation period for loading can be shortened by“stressing” the cells by, for example, omitting glucose or fetal bovineserum from the incubation medium. This increases the rate ofendocytosis, and accordingly shortens the time needed for the trehaloseor other disaccharide to be taken up. In these embodiments, the cellsare stressed and the incubation is for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 hours. Incubations of 3 to 20hours are generally preferred, with incubations of about 4 to 15 hoursbeing more preferred and 10 to 12 hours being more preferred.

The incubation is typically performed at 30 to 39° C., but is preferablyat the temperature considered normal for the mammal from which the cellsbeing incubated originated. For human cells, a temperature of 37° C. ispreferred. Preferably, the cells are incubated under tissue cultureconditions. Tissue culture conditions for maintaining cells of vanoustypes have been studied with some care and are known in the art. Forhuman cells, we prefer to incubate the cells in 5% CO₂ at 90% relativehumidity. Detailed information about tissue culture conditions foranimal cells, as well as media suitable for such cultures, can be foundin a number of sources, such as R. Freshney, Culture of Animal Cells,Wiley-Liss, New York (3rd Ed. 1994).

Trehalose has traditionally been the most preferred disaccharide forprotecting cells, in part because of its high glass transitiontemperature, and in part because its glycosidic bond is resistant todegradation in the endosome. The methods of the invention, however, donot require the disaccharide to have a high glass transitiontemperature, and we have found that sucrose, for example, can survivethe endosome. Accordingly, while trehalose is particularly preferred, itis believed that other disaccharides, such as sucrose and maltose, canbe used in place of trehalose in the methods of the invention.

The disaccharide should be present in the growth medium at between 50 to200 mM. We have found that about 80 to 150 mM is satisfactory in loadingtrehalose into cells of various tissues (e.g., MSCs and epithelialcells), with 90 to 120 mM being better and about 100 mM being preferred.Cells that circulate in the blood, such as B cells, load better atsomewhat lower concentrations of trehalose, with 50 to 100 mM beingsatisfactory, 60 to 85 mM being better, and about 75 mM being preferred(in the context of loading trehalose or other disaccharides, “about”means 5 mM plus or minus).

B. Use of DMSO to Improve Cellular Distribution of Trehalose

We have found that MSCs and epithelial cells load trehalose evenly.Different cell types however, may vary in their ability to distributetrehalose evenly, due perhaps to such factors as having a higher levelof membrane-bound proteins. The ability of a cell type to distributetrehalose can be estimated by such techniques as by the lucifer yellowassay discussed in the Examples. Lucifer yellow (more usually “Luciferyellow CH,” or “LYCH,” in recognition of a carbohydrazide moiety thatallow the molecule to be fixable with an aldehyde fixation agent) is acommercially available (from, e.g., Biotium, Inc. Hayward, Calif.)fluorescent dye which is of similar molecular weight and polarity totrehalose. Accordingly, it is assumed that LYCH provides anapproximation of how trehalose distributes within a cell type. The LYCHcan be assayed by fluorescence spectroscopy to determine overall uptake.Fluorescence microscopy can also be used to determine distribution. Thatis, if the LYCH is still contained within the endocytotic vesicles, thestaining appears punctate, but if the dye is distributed evenly, thestaining appears diffuse and homogeneous. If distribution into specificsubcellular compartments (e.g. mitochondria) is of concern, that can bedetermined by cell fractionation.

We have found that DMSO is not necessary for drying and rehydration ofcells, but it does improve intracellular distribution of trehalose. Itis believed that the trehalose stabilizes proteins and membranes duringthe drying process. Therefore, it is believed that any portions of thecell in which trehalose is absent would be more likely to sustain damageduring drying and rehydration, possibly comprising viability of thecell. If the cells are not distributing trehalose evenly, thereforedimethyl sulfoxide (“DMSO”) may be added. The DMSO may be added aslittle as 20 minutes before the end of the contemplated trehaloseloading time (that is, the time the cells are incubating in atrehalose-containing solution to load them with trehalose), morepreferably 30, 40, 50 or 60 minutes before the end of the trehaloseloading time. The DMSO may be added 2, 3, 4, 5, or 6 hours before theend of the trehalose-loading time. The DMSO may be added in amounts sothat the DMSO constitutes between 0.1% and about 2% DMSO by volume.

The lower limit of DMSO that is suitable can be determined byintroducing an amount (for example, 0.5% of DMSO) into the media,incubating for 2 hours, performing the lucifer yellow assay, andvisually observing if the dye is evenly distributed. If the dye is notevenly distributed, then the test is run on a parallel group of cellswith a higher percentage of DMSO to see if that provides adequatedistribution.

Amounts higher than 3% are not desirable since DMSO itself becomes toxicto cells at higher concentrations. Thus, providing the DMSO atpercentages of about 2% or below is preferred. Whether any particularamount between 2% and 3% is too toxic for the cells can be readilydetermined by introducing the amount into the media, incubating cells inthe media for 2 hours, and determining the percentage of viable cells bystandard cell viability assays, such as by taking a sample of the cells,diluting the sample with trypan blue and counting the viable (unstainedcells) and non-viable (stained cells) on a hemocytometer under amicroscope. The number of viable cells counted, divided by the totalnumber of cells, times 100, provides the percentage of viable cells.Percentages of DMSO which result in viability of less than 50% of thecells should be avoided. More preferably, percentages of DMSO are usedthat result in viability of 60%, 70%, 80% of the cells or higher.

It is noted that, following the trehalose loading incubation, the cellsare placed in a drying buffer, which does not contain DMSO. Thus, thebuffer in which the cells are dried; and in which they are subsequentlyrehydrated does not contain DMSO. Further, it is believed that theamount of DMSO taken into the cells given the modest amounts added tothe incubation buffer is small. Thus, the methods of the invention avoidthe toxicity problems which have been associated with DMSO's use as acryoprotective agent.

C. Use of Arbutin

Surprisingly, we have found that the ability of some cell types tosurvive being dried and then rehydrated is significantly enhanced byincluding the compound arbutin in the incubation medium. Arbutin (CASNumber 497-76-7, Beilstein Registry Number 89673), is also known ashydroquinone-beta-D-glucopyranoside, 4-hydroxyphenyl-beta-D-pyranoside,p-arbutin, and arbutine. It has the molecular formula C₁₂H₁₆O₇, and amolecular weight of 272.25. Arbutin was originally extracted from theleaves of plants such as Arctostaphylos uva-ursi (“bearberry”), and the“resurrection plant” Myrothamnus flabellifolia; natural and syntheticarbutin are now commercially available from a number of sources,including Sigma-Aldrich (St. Louis, Mo.); Kraeber GmbH & Co. (EllerbekGermany); Thinker Chemical Co., Ltd., (Hangzhou, China), Peakchemdivision, City Pride Co., Ltd. (Hangzhou, China), and Shanghai UCHEMCo., Ltd. (Shanghai, China). Arbutin is used commercially in topicalcosmetic agents for whitening skin.

As reported in the Examples, below, arbutin was found to enhance themetabolism of MSCs after drying and rehydration, and to enhance theability of MSCs to divide and differentiate after drying andrehydration. Interestingly, arbutin also induced the expression of heatshock protein 70 (HSP 70) in MSCs. The biological effects of arbutinwent beyond those expected from the induction of the HSP alone.Therefore, while the induction of HSP70 may be responsible for some ofthe effects observed, it is believed that the beneficial effect of thearbutin on cell metabolism and division is due to factors other than orin addition to the induction of the HSP alone.

As noted, arbutin is particularly useful in connection with stem cells,such as MSC, that are dried and then rehydrated. It can be added to theincubation medium preferably during the entire incubation withtrehalose, but can be added part way through the incubation if desired.It is desirable that it be present for at least 15 minutes, morepreferably, 30 minutes, still more preferably, 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more hours, with longer times being more preferred to permitthe cells to take in the arbutin. It can be present at 5 to 150 mM, with10 to 100 mM being more preferred, 20 to 80 mM being still morepreferred and 30 to 60 mM more preferable. We have found 40 mM to besatisfactory for loading stem cells, and use that as our preferredamount.

When arbutin is present in the incubation medium, the amount oftrehalose in the medium can be reduced. In the absence of arbutin, 100mM of trehalose is the preferred concentration in the incubation mediumfor loading stem cells. With arbutin present in the medium, theconcentration of trehalose can be reduced to 70 mM for stem cells.

Based on our studies, however, we believe the use of arbutin willimprove the viability of many cell types. It is, however, toxic to 393and B cells and therefore if used in loading arbutin-sensitiveepithelial cells or B cells, it should not be present at more than about10 mM. Whether arbutin is toxic or beneficial to preserving cells of anyparticular cell type can be readily determined by standard assays, suchas the trypan blue viability assay described above or the propidiumiodide (PI) exclusion assay described in the Examples.

As may be apparent from the chemical names for arbutin, it is aglycosylated hydroquinone. Thus, while arbutin in particularly preferredin the methods and compositions of the invention, it is believed thathydroquinone can be substituted for arbutin in the methods andcompositions described herein.

D. Heat Shock Proteins

We have further surprisingly found that the presence of heat shockproteins (“HSPs”, also known as “stress proteins”) in the cells prior todrying them enhances the viability of the cells upon rehydration. Theproteins can be endogenously produced in the cells in response to heatshock, can be exogenously provided, or can be expressed as a result oftransfecting the cells with a nucleic acid sequence encoding an HSP ofchoice.

As used herein, a “stress protein,” also known as a “heat shock protein”or “HSP,” is a protein that is encoded by a stress gene, and istherefore typically produced in significantly greater amounts upon thecontact or exposure of the stressor to the organism. A “stress gene,”also known as “heat shock gene” is used herein as a gene that isactivated or otherwise detectably upregulated due to the contact orexposure of an organism (containing the gene) to a stressor, such asheat shock, hypoxia, glucose deprivation, heavy metal salts, inhibitorsof energy metabolism and electron transport, and protein denaturants, orto certain benzoquinone ansamycins. Nover, L., Heat Shock Response, CRCPress, Inc., Boca Raton, Fla. (1991). “Stress gene” also includeshomologous genes within known stress gene families, such as certaingenes within the HSP70 and HSP90 stress gene families, even though suchhomologous genes are not themselves induced by a stressor. Each of theterms stress gene and stress protein as used in the presentspecification may be inclusive of the other, unless the contextindicates otherwise.

In preferred embodiments, the cells are briefly heated to a temperaturethat induces expression of heat shock proteins (that is, the cells are“heat shocked”). Heat shocking has been conducted on cells of manyspecies to study the effect of HSPs and patterns of HSP expression.Accordingly, the temperatures at which to shock cells of many mammalianspecies can be found in the literature or readily determined followingart-recognized techniques. For human cells, raising the cells (or themedium in which the cells are bathing) to a temperature of about 42-44°C. is preferred. At temperatures over 44° C., the viability of the cellsbegins to drop. The cells can be exposed to a quick pulse of heat, orthe temperature of the medium can be gradually stepped up. A quick pulseconsists of heating the medium containing the cells to the desiredtemperature for 20 minutes to 2 hours, with 1 hour being preferred. Fornon-human cells from animals with normal body temperatures higher thanthat of humans, correspondingly higher temperatures are useful to heatshock the cells. It can readily be determined whether any particulartemperature is too hot by performing a trypan blue assay as describedabove. Death of more than 20% of the cells indicates that too high atemperature has been used.

Following the heat shock, the temperature of the cells (or, moreprecisely, of the medium comprising the cells) is allowed to drop,usually back to the same temperature as that at which the cells werebeing incubated or grown prior to the shock, and permitted 10-48 hours,more preferably 20-28 hours, most preferably 24 hours, to express heatshock proteins induced by the heat shock. If desired, trehalose can beintroduced into the medium before the heat shock, during the heat shock,or following the heat shock during the period the cells are expressingthe heat shock proteins, to permit the cells to become loaded withtrehalose at the same time the heat shock proteins are being expressed,to reduce the overall time of the procedure, or trehalose can beintroduced into the medium after the heat shock protein-induction periodto load the cells with trehalose at that point.

The induction of HSPs can be confirmed by incubating the cells for 10-48hours to give them time to express the induced HSPs, lysing the cells,running the cell contents on a gel to separate the proteins,transferring the proteins from the gel to a blot (e.g,, nitrocelluloseor PVDF membrane) and probing the membrane with antibodies to HSPproteins. All of these procedures for determining the presence ofproteins are well known in the art. Antibodies to numerous HSP proteinsare commercially available. For example, Upstate Biotechnology, LakePlacid, N.Y., sells antibodies to HSP 27 and 90, while antibodies to HSP25, 27, 56, 70, 73, 84, 86, and 104 are available from Abcam Ltd.(Cambridge, UK). (Heat shock proteins are commonly referred to by theirmolecular weight expressed in kiloDaltons, or “kDa”). In studiesunderlying the present invention, heat shocking of murine B cellsresulted in the induction of HSP70.

As an alternative to inducing HSP expression in the cells, they can beincubated in a medium containing one or more HSPs of choice. It isanticipated that the cells will endocytose some amount of the HSPsduring the incubation. If it appears that the HSP is not beingendocytosed into the cells or that the HSP is entering the cells moreslowly than desired by the practitioner, the amount of uptake can beincreased by use of a protein delivery reagent, such as the BioPORTER®Quikease™ system described infra.

The cells can also be transfected with a vector encoding a heat shockprotein. Numerous HSPs have been studied since at least the 1980's andtheir amino acid and nucleic acid sequences are known. See, e.g., Hunt,C. and Marimoto, R. Proc. Natl. Acad. Sci. USA 82:6455-6459 (1985);Drabent, B. et al., Nucleic Acids Res. 14(22): 8933-8948 (1986);

Uoshima, K., et al., Biochem. Biophys. Res. Commun. 197(3):1388-1395(1993) (rat HSP27); Carper et al., Nucleic Acids Res. 18 (21): 6457(1990) (human HSP27) GenBank accession number NM 212504 (rat HSP70). Thestudies set forth in the Examples show the results of transfectingmammalian cells with a nucleic acid encoding a brine shrimp HSP, p26, asan exemplar HSP. Based on the results seen with using p26, we believethat any of the heat shock proteins of roughly 104 kDa or smaller willwork to increase viability of cells undergoing drying and rehydration.As noted, p26 is a brine shrimp protein, and protects human cells duringdrying and rehydration, as shown in the Examples. Accordingly, the HSPprotein does not have to be a human HSP, or even a mammalian HSP.

Persons of skill will be aware that some HSPs are constitutivelyexpressed, while others are induced when the cells are exposed to stressconditions or have their expression markedly increased under stressconditions. For purposes of the methods of the invention, HSPs that areinduced under stress conditions or which have their expression increasedwhen the cells are placed under stress are preferred, and HSPs that areinduced when cells are placed under stress conditions are particularlypreferred. As shown in the Examples, the HSP referred to as p26 protectshuman cells and is preferred for uses in which transfected cells aresuitable.

It will be appreciated that numerous vectors and promoters are known inthe art. The choice of the particular vector or promoter is not criticalto the invention. Since it is desirable that the HSP chosen be expressedin the cells to be dried, the promoter of course should be one that will“drive” expression of the protein under the conditions of the culture.Thus, it is preferred if the coding sequence for the protein be placedunder the control of a promoter that will either be constitutivelyactive or that will be active under the culture. In a preferredembodiment, the promoter is the human cytomegalovirus immediate-earlypromoter/enhancer. The CMV promoter/enhancer permits efficient,high-level expression of the recombinant protein in transfected cells.Expression of recombinant HSPs are known in the art. See, e.g., Li etal., Infect Immun. 69 (5): 2878-2887 (2001). Vectors and methods fortransfecting cells with HSPs, by themselves or in conjunction with otherproteins, are taught, for example, in U.S. Pat. No. 6,495,347.

Although transfected cells can be used in clinical applications,induction of endogenous HSPs is preferred, since it is more difficult toget regulatory approval to introduce into patients cells that have beenrecombinantly engineered. Thus, for example, HSPs for cells contemplatedfor therapeutic applications, such as MSC, are preferably induced byheat shock or are loaded into the cells from the medium. For in vitrouse, the HSPs can be endogenous or can be introduced by transfection.

In the studies reported in the Examples, human 293 epithelial cells weretransfected with p26. p26 was seen to provide a protective effect oncell survival and recovery upon rehydration after drying. Further, p26acted to inhibit apoptosis of the cells during drying. HSPs have alsobeen found to improve viability following drying and rehydration of HeLacells, and to decrease the presence of damaging reactive oxygen species(ROS) in HeLa cells during drying. HeLa cells are a cell line originallyderived from a cervical cancer; cervical cancers are considered to be ofepithelial origin. As noted above, arbutin is toxic to epithelial cells,and its use with epithelial cells is not preferred. Therefore, HSPs areparticularly useful in connection with epithelial cells.

E. Apoptosis Inhibitors

We have further surprisingly found that introducing one or moreapoptosis inhibitors into the incubation medium significantly enhancesthe viability of cells undergoing drying and then rehydration.Typically, the inhibitors interfere with caspases that are known to beinvolved in the apoptotic pathway. Four apoptosis inhibitors have beentested, and three were found to enhance survival of cells undergoingdrying and rehydration:

(a) N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone,(C₂₆H₂₅N₃O₆F₂). This compound is commercially available from MPBiomedicals (Irvine, Calif.) (MP Biomedicals, Calbiochem (San Diego,Calif.), Kamiya Biomedical Co. (Seattle, Wash.), and Imgenex (San Diego,Calif.) is a cell permeable, irreversible pan-caspase inhibitor;especially active against caspases 1, 3, 8, and 9. The compound iscommercially sold as as “Q-VD-oPh” or, in MP Biomedicals parlance,“OPH-109”, which name is used in the Examples. The term Q-VD-OPH (or“Q-VD-oPh”) denotes that the compound has a quinoline derivative (Q), adipeptide, valine (V, in standard single letter code) and aspartic acid(D, in standard single letter code), and a non toxic 2,6-difluorophenoxymethylketone (OPH) group. See, Caserta et al., Apoptosis 8(4): 345-352(2003); Rebbaa, A. et al. Oncogene 22:2805 (2003); Melnikov, et al., JClin Invest. 110: 1083-1091 (2002); Patil and Sharma, NeuroReport15:981-984 (2004). The mechanism of action involves the formation of anirreversible thioether bond between the aspartic acid derivative in theinhibitor and the active site cysteine of the caspase with thedisplacement or the 2,6-difluorophenoxy leaving group. According to MPBiomedicals literature, Q-VD-OPH is effective in vitro at concentrationsof 10 μM to 20 μM. For tissue culture studies 10 mM or 20 mM stocksolutions are prepared in DMSO and diluted 1:1000 directly into thetissue culture medium. For in vivo use, Q-VD-OPH has been administeredin 80% to 100% DMSO to assure solubility at the doses given. A dose of20 mg/kg has been used most frequently, but doses of 120 mg/kg have beenused in vivo studies. To reduce hydrophobicity, several of the suppliersmentioned above, such as Calbiochem, sell a version of Q-VD-oPh in whichthe aspartyl residue is not o-methylated.

(b) Caspase I inhibitor II (IL-1β Converting Enzyme (ICE) Inhibitor II,available from EMD Biosciences, Inc., San Diego, Calif.) acell-permeable and irreversible inhibitor of caspase-1 (Ki=760 pM) andcaspase-4, that inhibits Fas-mediated apoptosis and acidicsphingomyelinase activation;

(c) Calpain inhibitor (OXIS International, Inc., Portland, Oreg., see,e.g., Shinohara, K. et al., Biochem. J. 317:385 (1996)), acell-permeable inhibitor of calpain I (Ki=190 nM), calpain II (Ki=220nM), cathepsin B (Ki=150 nM, and cathepsin L (Ki=500 pM); and

(d) Bcl-xL (Biosource International, Camarillo, Calif.), acell-permeable peptide that prevents apoptotic cell death by directlybinding to the voltage-dependent anion channel (VDAC) and blocking itsactivity. Leads to the inhibition of cytochrome c release and loss ofmitochondrial membrane potential (DYm).

Of these four, the inhibitor Q-VD-OPH was found to be the most effectiveat retaining cell viability, particularly of murine B cells, duringdrying and rehydration. Calpain inhibitor was tested only in conjunctionwith OPH-109 and it did not increase survival over the use of OPH-109alone; its effect by itself was not tested.

Based on the results with these apoptosis inhibitors, it is expectedthat most if not all inhibitors of apoptosis will be beneficial inenhancing cell survival of drying and rehydration. Whether or not anyparticular apoptosis inhibitor is effective at increasing cell viabilitycan be readily determined following the teachings of this disclosure,including the assays taught herein.

Preferred inhibitors are those that are cell-permeable, so that they canenter the cell from the incubating medium, although cells can also beloaded with the inhibitor by using a commercially available proteindelivery reagent, such as BioPorter®. Numerous inhibitors of apoptosisare known and are commercially available. Calbiochem alone (a brand nameof EMD Biosciences, Inc., San Diego, Calif.) sells some twentyinhibitors of various caspases.

It is known that some inhibitors are constitutively expressed and someare induced under stress conditions or are much more strongly expressedunder stress conditions. Inhibitors that are induced under stressconditions or are much more strongly expressed under stress conditionsare preferred in the methods of the invention.

The inhibitor is preferably present in the incubation medium at aconcentration from 5 μM to 150 μM. More preferably, it is present from10 μM to about 100 μM, more preferably from 15 μM to about 80 μM andstill more preferably from about 20 μM to about 60 μM. We have had goodresults using apoptosis inhibitors at a concentration of 30 μM, which isaccordingly the most preferred.

Interestingly, in the studies underlying the invention, trehalose wasseen to block apoptosis in murine B cells during drying and rehydration.It does not, however, inhibit apoptosis due to heat shock or generally,and therefore its anti-apoptotic effect appears specific to dehydration.Trehalose is, of course, a disaccharide and by definition is not a heatshock protein.

F. Drying Buffer

Following incubation with trehalose to load the cells, the cells areharvested and placed in a drying buffer. If necessary to harvest thecells, they may be trypsinized to release them from a surface on whichthey have been incubated. The cells are then gently spun to pellet them.The supernatant is removed and replaced with a drying buffer. The dryingbuffer comprises trehalose or other disaccharide used to load the cells,which is preferably present in a concentration higher than that used inthe incubation medium. The trehalose or other disaccharide is preferablypresent at from 100 to 200 mM, with 120 to 180 mM being preferred, and140 to 170 mM being more preferred. We have found 150 mM to besatisfactory, and this concentration is our most preferred.

If arbutin or hydroquinone has been used in the incubation medium, thanit is preferred that it also be present in the drying buffer. Thearbutin or hydroquinone is preferably present at from 20 to 150 mM, with40 to 120 mM being preferred, and 50 to 100 mM being more preferred. Wefind 70 mM of arbutin to be satisfactory, and this concentration is themost preferred.

The drying buffer preferably comprises a bulking agent to help separatethe cells. Albumin is a preferred bulking agent. Human cells do notappear to be particularly sensitive to the type of albumin present; forhuman cells, human serum albumin or bovine serum albumin are bothacceptable. We have found that cells of some species do not toleratealbumin of some other species. Accordingly, if the cells to be dried arefrom a non-human mammal, it is desirable to place a sample of the cellsin culture medium to which the albumin to be used has been added andobserve the cells to see whether they lyse. If they lyse, albumin fromanother source organism should be tested until one is found which doesnot cause lysis. Albumin from the same organism as that from which thecells originated will be compatible. Other polymers suitable for use asbulking agents are, for example, water-soluble polymers such as HES(hydroxy ethyl starch), polyvinyl alcohol, and dextran.

G. Drying the Cells

Nucleated mammalian cells normally cannot withstand being dried to bonedryness, contrary to claims made by some researchers. We find that thecells are preferably dried to 0.2 to 0.5 grams of residual water pergram of dry weight.

We have found that the cells can be dried in any of three ways. First,and most preferably, the cells can be dried by vacuum drying. In thisembodiment, the cells are preferably dried in 50 μL aliquots. Roundeddroplets are preferred to spreading the cells onto a surface. TheExamples report significantly better viability of cells that are driedin rounded droplets of drying buffer rather than thin films. The cellsare preferably dried at room temperature, with 20 to 25° C. being thepreferred temperature range. The cells are dried under a vacuum ofapproximately 3 inches of mercury.

Second, the cells can be air dried. In this embodiment, the cells arepreferably dried in 50 μL aliquots. Once again, rounded droplets ofcells in drying buffer are preferred to spreading the cells onto asurface as a thin film. They can be dried at room temperature. In thisembodiment, the cells are dried under a diffuse stream of dry air untilthey reach the desired range of dryness.

Third, the cells can be dried by freeze drying. In this embodiment, thecells are preferably dried in 50 μL aliquots, which is typicallyperformed in a 10 drop array. Freeze drying can result in reducing thewater content to levels below 0.2 grams per gram dry weight. To avoidthis, the system is preferably calibrated by freeze drying parallelsamples of the cells of choice in the drying buffer contemplated for usefor different period of time in the lyophilizer to be used to determinethe time points at which the cells will be dried to the desired residualwater content.

H. Storing and Rehydrating the Cells

Once dried, the cells are preferably stored at 4° C. Preferably, thecells are stored under vacuum in an air-tight container to preventexposing them to changes in ambient humidity.

When rehydrating the cells is desired, the cells can be placed directlyinto a growth medium standard for the cell type in question or thegrowth medium can be added directly to the container in which the cellsare stored. Optionally, the cells are prehydrated prior to placing themin medium by exposing them to humid air. We have found, however, that aprehydration step is not necessary when rehydrating nucleated cellsdried according to the methods herein because of the residual watercontent of the cells. Since prehydration adds a step withoutcorresponding benefit, it is usually omitted.

The medium can be at room temperature or preferably from 25 to 39° C.(the temperature of the medium may be that normal for the species fromwhich the cells originate if it is higher than 39° C.). Preferably, theconditions of gases, temperature, and humidity under which the cells arerehydrated are those under which the cells were loaded with trehalose.

III. Uses

Cells dried by the methods of the invention can be convenientlytransported, preferably under refrigeration, Preferably, the transportis under vacuum in an air-tight container. Preferably, the container isalso water-impermeable to reduce premature, inadvertent rehydration ofthe cells in the event of encountering high humidity conditions oraccidental exposure to liquids during transport. The invention thereforeprovides a means for convenient shipping of dried cells, while reducingor eliminating the need for costly courier services and the specialhandling required for the liquid nitrogen-frozen cells of conventionaltechniques for preserving nucleated mammalian cells.

Cells dried and then rehydrated by the methods of the invention can beused for a number of applications. It is envisaged that epithelial cellsand other cells can be used in place of conventional cells in biosensorsto detect toxic substances in assays. It is further contemplated thatstem cells, such as ESCs, CBSCs, or MSCs, dried according to theinvention can be shipped to a location in need of such cells,rehydrated, and introduced into patients who can benefit from thepresence of such cells.

IV. Definitions

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety. Terms not defined herein have their ordinary meaning asunderstood by a person of skill in the art.

“Mammalian” means from a mammal. Canine, feline, equine, bovine andprimate mammals are preferred with humans being particularly preferred.

“Nucleated” refers to a cell that have a nucleus. It does not refer tocells such as red blood cells, that had a nucleus, but lost the nucleusin the course of differentiation, but does refer to red blood cellprecursors still in possession of a nucleus, or to blood platelets.

“293 cells” are a cell line of human embryonic kidney (“HEK”) cells.

“Cells of the immune system” refers to B cells, T cells, and dendriticcells.

“Arbutin” (CAS Number 497-76-7, Beilstein Registry Number 89673), is acompound also known as hydroquinone-beta-D-glucopyranoside,4-hydroxyphenyl-beta-D-pyranoside, p-arbutin, and arbutine. It has themolecular formula C₁₂H₁₆O₇, and a molecular weight of 272.25.

“Exogenous,” when referring to the presence of heat shock proteins orapoptosis inhibitors in a cell of a given type, means a heat shockprotein or apoptosis inhibitor not expressed by a normal cell of thattype. For example, the heat shock protein p26 from Artemia franciscanais not expressed by human cells unless they have been altered by beingtransfected with a nucleic acid sequence encoding p26. The term isintended to distinguish heat shock proteins or apoptosis inhibitorsintroduced from outside the cell, or with which the cell is transfected,from heat shock protein or apoptosis inhibitors the cell might naturallyexpress in response to environmental change or in response to signalingfrom other cells.

“Contacting” means bringing into physical contact.

V. Heat Shock Proteins

Heat shock proteins (HSP) are considered to be stress proteins. HSPsassist the folding of proteins, reduce stress-associated proteindenaturation and aggregation, aid in renaturation, and influence thefinal intracellular location of mature proteins. Stress proteins aredivided into groups or families, including Hsp100, Hsp90, Hsp70, Hsp60(the chaperonins), and the small heat shock/α (alpha) crystallinproteins, sometimes referred to as α-Hsps. Small heat shock proteinsincluding α (alpha) crystallin proteins are low molecular weight heatshock proteins, ranging in size from about 10- to 40-kDa monomermolecular mass, but oli-gomerize into particles of varying monomernumbers. The functions of chaperones differ, but their activities areinterrelated and often dependent on association into macromolecularcomplexes, sometimes consisting of representatives from more than onegroup or family.

P26 belongs to the α crystallin or α-Hsps group or family. As with manyother Hsps, α-Hsps or a crystallin proteins protect cells during stressby preventing aggregation of unfolded proteins and in some casesassisting in their renaturation. As indicated, p26 is a small heatshock/α crystallin protein, and has a diameter of about 15 nm, or about520 kDa It has 28 subunits, each being about 20.7 kDa. When biologicalsamples are nucleated cells, stress causes p26 to move into the nucleusof the nucleated cell.

P26 is found in the encysted embryos of the primitive crustacean Artemiafranciscana (the North American brine shrimp). Encysted embryos of A.franciscana contain very large amount of the α-Hsp or p26, making upfrom about 12% to about 15% by weight of the total nonyolk protein. Theremarkable stress resistance of Artemia cysts including p26 protectsshrimp embryo cells during encystment, diapause, and anaerobicquiescence, and prevents the aggregation of other proteins when shrimpembryos experience stresses of various kinds; thus playing an importantrole in their growth and development.

For a comprehensive discussion of p26, including procedures on purifyingp26 to homogeneity and measuring the concentration of p26, see Influenceof trehalose on the molecular chaperone activity of p26, a small heatshock/α-crystallin protein by Viner and Clegg., Cell Stress SocietyInternational, Cell Stress & Chaperones (6(2), pp. 126-135 (2001)). Foranother comprehensive discussion of p26, including the cloning andsequencing a cDNA for p26, the listing of the complete sequence ofp26-3-6-3 and the deduced amino acid sequence of p26, and the comparisonof the deduced amino acid sequence of p26 to other small heat shock/αcrystallin proteins (e.g., αA-crystallin, human αB-crystallin, humansmall heat shock protein 27 (H27), and a Drosophila small heat shockprotein known as embryonal lethal (2) 13-1 (Dro)), see MolecularCharacterization of a Small Heat Shock/alpha-Crystallin Protein inEncysted Artemia Embryos by Liang et al., J Biol Chem (272(30):19051-19058 (1997)). See also, Liang et al., Purification, structure andin vitro molecular-chaperone activity of Artemia p26, a smallheat-shock/alpha-crystallin protein, Eur. J. Biochem. 243 (1-2): 225-232(1997). The protein sequence of p26 may be obtained from the NationalCenter for Biological Information (NCBI) website under accession numberAAB87967. The cDNA sequence coding for p26 is available from theNational Center for Biological Information (NCBI) under accession numberAF031367.

The Hsp70 family is a multi-gene family of chaperones but all membershave four common features: highly conserved sequence, molecular massabout 70 kDa, ATPase activity and an ability to bind and release ofhydrophobic segments of unfolded polypeptide chains. The protein knownas Hsp 70, however, is the only member of the family that is stronglyinducible by heat stress.

In an embodiment of the invention, the p26 gene (p26 cDNA) is ligatedinto the vector (such as pSecTag2A DNA) by use of T4 DNA ligase and thencloned in Escherichia coli DHSa. The p26-containing plasmid produced byligation may be mixed in a tube and incubated at room temperature for asuitable period of time (e.g., 5 to 30 minutes) with an agent thatenhances transfection before application to the biological sample(s) fortransfection. For example, Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) is a cationic lipid-based transfection reagent. Othertransfection reagents are known and may be used in place ofLipofectamine 2000 which will, however, be mentioned as an exemplarreagent herein. The vector may be placed in a culture solution, suchas-serum-free DMEM (Dulbecco's Modified Eagle Medium), to produce atransfecting solution.

For transfecting, the biological sample(s) may be treated with thetransfecting solution in any suitable manner, such as by immersing thecells in the transfecting solution for a suitable period of time (e.g.,10 to 50 minutes). In an embodiment of the invention, transfections ofcells was accomplished by the use of 800 nanograms of the plasmid DNAmixture and 3 μd of Lipofectamine 2000 in 60 μl of serum-free DMEM forcells in each well of a six-well culture plate. It is to be understoodthat the cells may be transfected with the stress protein before beingloaded with the solute, after being loaded with the solute, orsimultaneously with the loading of the solute.

VI. Aspects of Loading

In some embodiment of the invention, the stress protein maybe loadedinto the biological sample(s) (i.e., into non-transfected biologicalsample(s)) by any suitable means and/or method(s), such as by theemployment of a protein-loading solution (e.g., a p26-loading solution).For this embodiment of the invention, the stress protein, preferably thestress protein in essentially pure form, would be mixed with a suitableprotein-loading solution (e.g., a p26-loading solution), and thebiological sample(s) would subsequently be disposed in theprotein-loading solution for causing the transfer of the stress proteinfrom the protein-loading solution into the biological sample(s). Theprotein-loading solution may be any suitable physiologically acceptablesolution in an amount and under conditions effective to cause uptake or“introduction” of the stress protein from the protein-loading solutioninto the biological sample(s). Broadly, by way of example only, aphysiologically acceptable solution comprises one or more of thefollowing: the stress protein (e.g., p26), a salt solution (e.g., PBS),a protein (e.g., BSA), and a carbohydrate (e.g., a starch, anoligosaccharide, etc). In other embodiments of the invention, thephysiologically acceptable solution comprises one or more of thefollowing: the plasmid DNA mixture (e.g., the plasmid DNA mixture),Lipofectamine 2000, a salt solution (e.g., PBS), a protein (e.g., BSA),and a carbohydrate (e.g., a starch, an oligosaccharide, etc).

The loading temperature of the protein-loading solution for loading astress protein into the biological sample(s) may be any suitabletemperature, such as a temperature ranging from about 25 degrees C. toabout 60 degrees C., more preferably from about 30 degrees C. to about40 degrees C., more preferably yet from about 36 degrees C. to about 38degrees C. The loading/incubating time for loading the stress proteinmay be any suitable time, such as a time ranging from about 10 minutesto about 48 hours, more preferably from about 30 minutes to about 34hours, most preferably from about 45 minutes to about 24 hours.

In another embodiment of the invention the stress protein may bedelivered into the biological sample(s) through the use of a proteindelivery kit sold as the BioPORTER® Quikease Protein Delivery Kit 2(Sigma-Aldrich Corp., St. Louis, Mo.). Suitable BioPORTER® proteindelivery kits are sold under product Nos. BPQ24 and BPQ96. TheBioPORTER® delivery kit has a BioPORTER® reagent which reacts quicklyand interacts non-covalently with the stress protein (e.g., p26) forcreating a protective vehicle for immediate delivery into biologicalsample(s). In embodiments of the invention the BioPORTER® reagent isincubated with the stress protein (e.g., p26) for a suitable period oftime, such as from about 2 mins. to about 15 mins.(e.g., 5 mins.).Subsequently, the resulting incubated product having the stress proteinis then incubated with the biological sample(s) for 1 to 8 hours (e.g.,about 4 hours). In an embodiment of the invention, the BioPORTER®reagent-stress protein complex is taken up by fluid phase endocytosis,subsequently fusing with a membrane (e.g., an endosome membrane) of thebiological sample(s) and releasing the stress protein into thecells(e.g., into the cytosol of the cells). The foregoing procedure mayalso be employed for loading the solute simultaneously with the loadingof the stress protein.

Embodiments of the present invention will be explained by loading of thesolute into the biological sample(s) after the biological sample(s)contain the stress protein. However, it is to be understood that thespirit and scope of the present invention includes loading ortransfecting the biological sample(s) with the stress protein after thebiological sample(s) is/are loaded with the solute, or simultaneouslywith the loading of the solute. Thus, embodiments of the invention arenot to be restricted to any particular order with respect to loading, ortransfecting, the biological sample(s) with the stress protein, and theloading of the solute into the biological sample(s). The loading, ortransfecting, the biological sample(s) with the stress protein may be:(i) before the biological sample(s) is/are loaded with the solute; (ii)after the biological sample(s) has/have been loaded with the solute; or(iii) simultaneously with the loading of the solute.

After the biological sample(s) has/have been transfected with/by, orhas/have been loaded with, a desired amount of the stress protein (e.g.,p26), the biological sample(s) may then be loaded with a suitablesolute. Broadly, the preparation of solute-loaded biological sample(s)containing the stress protein in accordance with embodiments of theinvention comprises the steps of loading one or more biologicalsample(s) with a solute by placing one or more biological sample(s) in asolute solution having a solute concentration of sufficient magnitudefor transferring the solute from the solution into the biologicalsample(s). For increasing the transfer or uptake of the solute from thesolute solution, the solute solution temperature or incubationtemperature has a temperature above about 25° C., more preferably above30° C., such as from about 30° C. to about 40°0 C.

The solute solution for various embodiments of the present invention maybe used for loading and/or drying and/or rehydration, or for any othersuitable purpose. When the solute solution is employed for loading asolute into the biological sample(s), the solute solution may be anysuitable physiologically acceptable solution in an amount and underconditions effective to cause uptake or “introduction” of the solutefrom the solute solution into the biological sample(s). Aphysiologically acceptable solution is a suitable solute-loading buffer,such as any of the buffers stated in the previously mentioned relatedpatent applications, all having been incorporated herein by referencethereto. The solute solution may also be any suitable physiologicallyacceptable solution in an amount and under conditions effective fordrying and/or rehydration. Therefore, the solute solution may be used asa drying buffer for drying loaded biological sample(s) and/or as arehydration buffer for rehydrating biological sample(s) inreconstituting biological sample(s). Thus, any of the solute solutionsfor embodiments of the present invention may be used for any suitablepurpose, including loading, drying, and rehydration.

For particular embodiments of the present invention, especially when thesolute solution is being employed as a loading buffer, the solutesolution comprises a solute (e.g., 50 mM to 150 mM trehalose) and a saltsolution (e.g., such as PBS). In other particular embodiments of theinvention, especially when the solute solution is being employed as adrying buffer and/or a rehydration buffer, the solute solution comprisesone or more of the following: a salt solution (e.g., PBS), a protein, asolute and an acid (e.g., HEPES, or N-(2-hydroxyl ethyl)piperarine-N′-(2-ethanesulfonic acid)). However, it is to be understoodthat the solute solution comprising one or more of a salt solution, aprotein, a solute, and an acid may be used for any other suitablepurpose. An example of a growth medium would be DMEM.

The salt solution may be any suitable physiologically acceptablesolution in an amount and under conditions effective to function as acarrier medium for a solvent, or for a mixture of a solvent, a proteinand/or an acid. The salt solution may comprise KCl and NaCl, such asmore particularly about 1 to 15 mM KCl and about 40 to 80 mM NaCl withpH 7.2. The salt solution may also comprise a phosphate buffered saline(PBS) solution comprising NaCl, Na₂HPO₄, and KH₂PO₄. A suitable PBSbuffer comprises a buffer sold under the product name Dulbecco's PBS(DPBS, Gibco cat # 14190), or a buffer comprising 283 mOsm PBS buffer(NaCl, Na₂HPO₄, KH₂PO₄, pH 7.2).

The acid may be any suitable acid. Preferably, the acid comprises asulfonic acid, such as, by way of example only, 5 to 20 mM HEPES(N-(2-hydroxyl ethyl) piperarine-N′-(2-ethanesulfonic acid)).

The carbohydrate for various embodiments of the invention is preferablytrehalose. The amount of the preferred trehalose loaded inside thebiological sample(s) ranges from about 10 mM to about 60 mM (e.g., up toabout 50 mM), and is achieved by incubating the biological sample(s) topreserve biological properties during drying with a trehalose solution.The effective loading of trehalose is also preferably accomplished bymeans of using an elevated temperature of from greater than about 25° C.to less than about 40° C. more preferably from about 30° C. to less thanabout 40° C., most preferably about 37° C.

In an embodiment of the invention where the solute is to be loaded intothe biological sample(s) simultaneously with the loading of thebiological sample(s) with the stress protein, the solute solutioncomprises the stress protein, the solute, a salt solution (e.g., PBS)and optionally one or more of the following: a protein (e.g., BSA), anda carbohydrate (e.g., trehalose).

The loading temperature of the solute solution for this embodiment ofthe invention ranges from about 25 degrees C. to about 40 degrees C.,more preferably from about 36 degrees C. to about 38 degrees C. Theloading/incubating time for loading the stress protein may be anysuitable time, such as a time ranging from about 10 hours to about 48hours, more preferably from about 18 hours to about 36 hours, mostpreferably from about 22 hours to about 24 hours.

Albumin may serve as a bulking agent, but other polymers may be usedwith the same effect. Suitable other polymers, for example, arewater-soluble polymers such as HES (hydroxy ethyl starch), polyvinylalcohol, and dextran.

The solute-loaded, stress protein-contained biological sample(s) in thedrying buffer may then be dried by the means described above, such as byvacuum drying, air drying, or freeze drying, all known in the art.Vacuum drying is the most preferred, with air drying less preferred tovacuum drying, and freeze drying least preferred.

The solute-loaded, stress protein-containing cells in the drying buffermay be vacuum dried in accordance with well known procedures. Biologicalsample(s) loaded with trehalose and producing p26 may be aliquotted intovolumes of 50-150 μL and subjected to vacuum in the range of 3 inches Hgat room temperature for a period in the range of 2 to 4 hours. Thisvacuum drying technique would bring the water content in the biologicalsamples) down to about 0.2 gm. H₂O/gm. dry weight.

The solute-loaded, stress protein-contained biological sample(s) in thedrying buffer may be air dried in accordance with well known procedures.Biological samples loaded with trehalose and producing p26 may bealiquotted into volumes of 50 uL-1.0 mL and dried either in a biohood orin a desiccator modified to distribute a stream of dry air evenly acrossthe surface of the biological sample(s). The drying may be conducted atroom temperature for a period in the range of 6 to 10 hours. This airdrying technique would bring the water content in the biologicalsample(s) down to about 0.2 g/m. H₂O/gM. dry weight.

If the solute-loaded, stress protein-contained biological sample(s) inthe drying buffer are freeze-dried, the solute-loaded, stressprotein-contained biological sample(s) in the drying buffer may be driedwhile simultaneously cooled to a temperature below about −32° C. Acooling, that is, freezing, rate is preferably between −30° C. and −1°C./min. and more preferably between about −2° C./min to −5° C./min.Drying may be continued until about 95 weight percent of water has beenremoved from the biological sample(s). During the initial stages oflyophilization, the pressure is preferably at about 10×10⁻⁶ torr. As thebiological samples dry, the temperature can be raised to be warmer than−32° C. Based upon the bulk of the biological samples, the temperatureand the pressure it can be empirically determined what the mostefficient temperature values should be in order to maximize theevaporative water loss. Dried (e.g., freeze dried) biological samplecompositions preferably have from 0.2 to 0.5% gram of water per gram dryweight.

The viability (e.g., the % viability) of biological sample(s) afterdrying may be determined by fluorescent live/dead analyses. There areseveral commercially available fluorescent live/dead kits. These kitswork on the same principles as trypan blue; that is, dead biologicalsample(s) with compromised plasma membranes will take upmembrane-impermeant dyes. A typical live/dead kit may contain a membranepermeant dye (e.g. syber green, SG, from Molecular Probes), which willstain all the biological sample(s), and a membrane-impermeant dye (e.g.propidium iodide, PI), which will stain only the dead biological.sample(s). The percentage of dead biological sample(s) is calculated bycounting the PI-stained biological sample(s) and dividing by theSG-stained biological sample(s). The percentage of viable biologicalsample(s) is calculated by subtracting the % dead biological sample(s)from 100.

After drying and storage of the biological sample(s), the process ofusing such a dehydrated biological-sample composition comprisesrehydrating the biological sample.

It has been discovered that the ability of dried biological sample(s)having a stress protein (e.g., p26) to proliferate and form coloniesafter rehydration is greater than the ability of dried biologicalsample(s) not having a stress protein to proliferate and form colonies.It has also been further discovered that the ability of dried biologicalsample(s) having a stress protein and a solute to proliferate and formcolonies after rehydration is also greater than dried biologicalsample(s) not having a stress protein, or having a solute and no stressprotein. The proliferation or the number of colonies formed by thebiological sample(s) after drying and rehydration may be determined byany suitable procedure well known to those skilled in the art. By way ofexample only, after rehydration, the biological sample(s) may be platedinto T-25 flasks and incubated at 37° C. for 7 days. The biologicalsample(s) may then be subsequently stained with either Coomassie blue orHema 3, and the colonies in each flask may be counted to obtain theproliferation or the number of colonies formed by the biologicalsample(s) after drying and rehydration.

Embodiments (e.g., the viability of dried biological sample(s),proliferation of biological sample(s) after drying and rehydration,etc.) of the present invention will be illustrated with eukaryotic 293cells (which are epithelial in origin) and by reference to FIGS. 1-10.It is to be understood that such use of 293 cells and such reference toFIGS. 1-10 are for exemplary purposes only, and are not to limit any ofthe embodiments of the present invention, or limit the spirit and scopeof the present invention in general.

FIG. 1 shows a graph of T-293 cell viability (%) subsequent to dryingvs. water content (gm. water/gm. dry weight) after drying fortransfected 293 cells (T-293 cells) and for control 293 cells(293-cells), with both the transfected 293 cells and the control 293cells having no trehalose internally and with the drying buffer for bothtransfected 293 cells and control 293 cells having no trehalose. ExampleII below provides the more specific testing conditions and parameterswhich produced the graphical illustrations of FIG. 1. Graph 10 and graph12 in FIG. 1 represents the transfected 293 cells, and the control 293cells, respectively. FIG. 1 illustrates that transfected T-293 cellstransfected with p26 survive drying better than control 293 cells nothaving been transfected with p26. Alternatively, FIG. 1 may illustratethat, when there is no trehalose inside or outside, there is nodifference in survival between the two types of cells.

FIG. 2 is a graph of viability (%) subsequent to drying vs. watercontent (gm. water/gm. dry weight) after drying for transfected 293cells (T-293 cells) and for control 293 cells (293-cells), with both thetransfected 293 cells and the control 293 cells having no trehaloseinternally and with the drying buffer for both transfected 293 cells andcontrol 293 cells having 150 mM trehalose. Example III below providesthe more specific testing conditions and parameters which produced thegraphical illustrations of FIG. 2. Graph 20 and graph 22 in FIG. 2represents the transfected 293 cells, and the control 293 cells,respectively. FIG. 2 illustrates that survival of transfectedT-293-cells is improved compared to control 293 cells when thetransfected T-293 cells are dried in a drying buffer having 150 mMtrehalose.

In FIG. 3 there is seen a graph of viability (%) subsequent to dryingvs. water content (gm. water/gm. dry weight) after drying fortransfected 293 cells (T-293 cells) and for control 293 cells(293-cells), with both the transfected 293 cells and the control 293cells having trehalose internally and with the drying buffer for bothtransfected 293 cells and control 293 cells having 150 mM trehalose.Graph 30 and graph 32 in FIG. 3 represents the transfected 293 cells,and the control 293 cells, respectively. Example IV below provides themore specific testing conditions and parameters which produced thegraphical illustrations of FIG. 3. FIG. 3 illustrates that mammalianT-293 cells transfected with p26, and loaded with trehalose, and driedin a drying buffer having trehalose greatly improves survival and/orviability when compared to control 293 cells not transfected with p26.

FIG. 4 is a graph of the number of colonies formed after rehydration vswater content after drying the transfected 293 cells (T-293 cells) andthe control 293 cells (293-cells) to 0.3 gm. water/gm. dry weight and to0.2 gm. water/gm. dry weight, with both the transfected 293 cells andthe control 293 cells having trehalose internally and with the dryingbuffer for both transfected 293 cells and control 293 cells having 150mM trehalose. Blocks 40 and 44 respectively represent transfected 293cells for water contents of 0.3 gm water/gm dry weight and to 0.2 gmwater/gm dry weight. Block 42 and the number “0” represented by numeral46 respectively represent control 293 cells for water contents of 0.3 gmwater/gm dry weight and to 0.2 gm water/gm dry weight. Example V belowprovides the more specific testing conditions and parameters whichproduced the graphical illustrations of FIG. 4. In the experiment thatproduced the results and graphical illustrations of FIG. 4, transfected293 cells and control cells were both dried respectively to 0.3 gmwater/gm dry weight and to 0.2 gm water/gm dry weight, rehydrated andthen plated (cultured) to determine their ability to form coloniessubsequent to rehydration (a measure of long-term proliferation andsurvival). As illustrated in FIG. 4, transfected T-293 cells dried to0.3 gm water/gm dry weight were able to produce colonies 20× greaterthan the control 293 cells. This pattern persisted at lower watercontents of 0.2 gm water/gm dry weight. However, no control 293 cellswere able to proliferate at a water content of 0.2 gm water/gm dryweight, while a significant fraction of the transfected T-293 cells didso.

Referring in detail now to FIG. 5, there is seen a graph of survival (%viability) vs. water content (gm. water/gm. dry weight) for a firstbatch of p26-transfected 293 cells (T-293 cells) after air drying andrehydration, and for a second batch of p26-transfected 293 cells (T-293cells) after vacuum drying and rehydration, with both batches of thep26-transfected 293 cells having trehalose internally. Example VI belowprovides the more specific testing conditions and parameters whichproduced the graphical illustrations of FIG. 5. Graph 50 and graph 52 inFIG. 5 represents vacuum-dried transfected 293 cells, and air-driedtransfected 293 cells, respectively. FIG. 5 broadly illustrates thatcell survival increases (e.g., increases by from about 20% to about 90%)by vacuum drying as opposed to air drying. FIG. 5 more specificallyillustrates that after p26 transfected T-293 cells were loaded withtrehalose (e.g., 25 mM to 800 mM trehalose) while incubating at atemperature above about 25° C. (e.g., from about 35° C. to about 40°C.), and then vacuum dried, instead of or as opposed to air drying,until the p26 transfected T-293 cells comprised a residual water contentof less than or equal to about 2.0 grams of water per gram of dry weightof T-293 cells, survival (% viability) increases. FIG. 5 also morespecifically illustrates that had the trehalose-loaded, p26 transfectedT-293 cells been air dried, instead of or as opposed to vacuum dried, tothe extent that the trehalose-loaded, p26 transfected T-293 cells had aresidual water content of greater than (or equal to) about 2.0 grams ofwater per gram of dry weight of T-293 cells, survival (% viability)increases. Thus, air drying is the preferred drying technique fortrehalose-loaded, p26 transfected T-293 cells if the residual watercontent of the trehalose-loaded, p26 transfected T-293 cells ismaintained at greater than (or equal to) about 2.0 grams of water pergram of dry weight of T-293 cells (e.g., from about 2.0 grams of waterper gram of dry weight of T-293 cells to about 8.0 grams water per gramof dry weight of T-293 cells); and vacuum drying is the preferred dryingtechnique for trehalose-loaded, p26 transfected T-293 cells if theresidual water content of the trehalose-loaded, p26 transfected T-293cells is maintained at less than (or equal to) about 2.0 grams water pergram of dry weight of T-293 cells. As shown in FIG. 5, the survival ofthe 293 cells (i.e., the biological sample) is preferably at least about60% (e.g., such as from about 60% to about 80%), more preferably atleast about 80%.

In FIG. 6 there is seen a graph of survival (% viability) vs. watercontent (gm. water/gm. dry weight) for a first batch of transfected 293cells (T-293 cells) after air drying while in a thin film configurationand after rehydration, and for a second batch of transfected 293 cells(T-293 cells) after air drying while in a plurality of dropletsconfiguration and after rehydration, with both batches of thetransfected 293 cells having trehalose internally. In FIG. 7 there isseen a graph of survival (% viability) vs. water content (gm. water/gm.dry weight) for a first batch of transfected 293 cells (T-293 cells)after vacuum drying while in a thin film configuration and afterrehydration, and for a second batch of transfected 293 cells (T-293cells) after vacuum drying while in a plurality droplet configurationand after rehydration, with both batches of the transfected 293 cellshaving trehalose internally.

A thin film configuration for the drying solution or buffer has a filmcontaining cells and has a thickness from about 0.1 mm to about 8.0 mm,preferably from about 0.50 mm to about 3.00 mm. A droplet or beadconfiguration for the drying solution or buffer contains cells and has abead or droplet physical configuration. When the loaded transfected 293cells (T-293 cells) are to be dried in a drying solution having roundeddroplets or beads, each droplet or bead would have an average volumeranging from about 10 μL to about 250 μL, preferably from about 20 μL toabout 150 μL, more preferably from about 30 μL to about 100 μL, mostpreferably from about 40μ to about 70 μL (e.g., about 50 μL).

Example VII below provides the more specific testing conditions andparameters which produced the graphical illustrations of FIG. 6 and ofFIG. 7. Graph 60 in FIG. 6 illustrates the viability (% viability)following rehydration of air-dried, rounded (i.e., bead-shaped) dropletsof drying solution containing trehalose-loaded transfected 293 cells.Graph 62 in FIG. 6 illustrates the viability (% viability) followingrehydration of an air-dried, thin film drying solution containingtrehalose-loaded transfected 293 cells. Graph 90 in FIG. 7 illustratesthe viability (% viability) following rehydration of vacuum-dried,rounded (i.e., bead-shaped) droplets of drying solution containingtrehalose-loaded transfected 293 cells. Graph 92 in FIG. 7 illustratesthe viability (% viability) following rehydration of a vacuum-dried,thin film drying solution containing trehalose-loaded transfected 293cells.

FIG. 6 broadly illustrates that cell survival increases (e.g., increasesby from about 20% to about 90%) by air drying rounded (i.e.,bead-shaped) droplets of drying solution containing trehalose-loadedtransfected 293 cells to a water content of less than or equal to about3 grams of water per gram of dry weight of T-293 cells, instead of airdrying in thin film configuration the drying solution containingtrehalose-loaded transfected 293. FIG. 6 also broadly illustrates thatwhen the drying solution containing trehalose-loaded, p26 transfectedT-293 cells are air dried in a thin film configuration (instead of or asopposed to a rounded droplet configuration) to the extent that thetrehalose-loaded, p26 transfected T-293 cells had a residual watercontent of greater than (or equal to) about 3.0 grams of water per gramof dry weight of T-293 cells, survival (% viability) of the 293 cellsincreases after rehydration. Thus, when air drying is the dryingtechnique for trehalose-loaded, p26 transfected T-293 cells, survival (%viability) is greatest if the drying solution containing thetrehalose-loaded, p26 transfected T-293 cells is in thin filmconfiguration and if the residual water content of the trehalose-loaded,p26 transfected T-293 cells is maintained at greater than (or equal to)about 3.0 grams of water per gram of dry weight of T-293 cells (e.g.,from about 3.0 grams of water per gram of dry weight of T-293 cells toabout 8.0 grams water per gram of dry weight of T-293 cells); androunded droplets configuration is the preferred configuration for thedrying solution containing trehalose-loaded, p26 transfected T-293 cellsif the residual water content of the trehalose-loaded, p26 transfectedT-293 cells is maintained at less than (or equal to) about 3.0 gramswater per gram of dry weight of T-293 cells. As shown in FIG. 5, thesurvival of the 293 cells (i.e., the biological sample) is preferably atleast about 600 (e.g., such as from about 60% to about 80%), morepreferably at least about 80%.

FIG. 7 broadly illustrates that cell survival increases (e.g., increasesby from about 5% to about 20%) by vacuum drying rounded (i.e.,bead-shaped) droplets of drying solution containing trehalose-loadedtransfected 293 cells to a water content ranging from a value greaterthan or equal to about 1 grams of water per gram of dry weight of T-293cells to a value less than or equal to about 3.5 grams of water per gramof dry weight of T-293 cells, instead of vacuum drying in thin filmconfiguration the drying solution containing trehalose-loadedtransfected 293. FIG. 7 also broadly illustrates that when the dryingsolution containing trehalose-loaded, p26 transfected T-293 cells arevacuum dried in a thin film configuration (instead of or as opposed to arounded droplet configuration) to the extent that the trehalose-loaded,p26 transfected T-293 cells had a residual water content ranging from avalue greater than (or equal to) about 3.5 grams of water per gram ofdry weight of T-293 cells to a value less than or equal to about 7.5grams of water per gram of dry weight of T-293 cells, survival (%viability) of the 293 cells increases after rehydration. Thus, whenrounded droplets are to be configuration for drying the drying solutioncontaining trehalose-loaded, p26 transfected T-293 cells, drying may beby either air drying or vacuum drying if the residual water content ofthe trehalose-loaded, p26 transfected T-293 cells is maintained at lessthan (or equal to) about 3.0 grams of water per gram of dry weight ofT-293 cells; and a thin film configuration is the preferredconfiguration for the drying solution containing trehalose-loaded, p26transfected T-293 cells if the residual water content of thetrehalose-loaded, p26 transfected T-293 cells is maintained at greaterthan (or equal to) about 3.0-grams water per gram of dry weight of T-293cells.

In FIG. 8 there is a graph of survival (% average viability) vs. watercontent (gm. water/gm. dry weight) for 293 cells (293 cells) aftervacuum drying while in a plurality droplet configuration and afterrehydration, and for transfected 293 cells (T-293 cells) after vacuumdrying while in a plurality droplet configuration and after rehydration,with both the 293 cells and the transfected 293 cells having trehaloseinternally (e.g., from about 25 mM to about 800 mM of internaltrehalose). Example VIII below provides the more specific testingconditions and parameters which produced the graphical illustrations ofFIG. 8. Graph 100 in FIG. 8 illustrates the viability (% averageviability) following rehydration of vacuum-dried, rounded (i.e.,bead-shaped) droplets of drying solution containing trehalose-loadedtransfected 293 cells. Graph 102 in FIG. 8 illustrates the viability (%average viability) following rehydration of a vacuum-dried, rounded(i.e., bead-shaped) droplets of drying solution containingtrehalose-loaded 293 cells (i.e., trehalose-loaded non-transfected 293cells). FIG. 8 broadly illustrates that cell survival increases (e.g.,increases by from about 10% to about 20%) by vacuum drying rounded(i.e., bead-shaped) droplets of drying solution containingtrehalose-loaded transfected 293 cell's to a water content of less thanor equal to about 5 grams of water per gram of dry weight of T-293 cells(e.g., from about 0.1 grams of water per gram of dry weight of T-293cells to about 5.0 grams water per gram of dry weight of T-293 cells),instead of vacuum drying rounded (i.e., bead-shaped) droplets of dryingsolution containing trehalose-loaded 293 cells (i.e., trehalose-loadednon-transfected 293 cells).

FIG. 9 sets forth a flow chart of a preferred embodiments of theinvention.

Embodiments of the present invention will be illustrated by thefollowing set forth examples which are being given to set forth by wayof illustration only and not by way of limitation. It is to beunderstood that all materials, chemical compositions and proceduresreferred to below, but not explained, are known to those artisanspossessing skill in the art. All materials and chemical compositionswhose source(s) are not stated below are readily available fromcommercial suppliers, which are also known to those artisans possessingskill in the art. Parameters such as concentrations, mixing proportions,temperatures, rates, compounds, etc., set forth in these examples arenot to be construed to unduly limit the scope of the invention.Abbreviations used in the examples, and elsewhere, are as follows:

DMSO=dimethylsulfoxide; ADP=adenosine diphosphate

PGE1=prostaglandin E1; HES=hydroxy ethyl starch

FTIR=Fourier transform infrared spectroscopy

EGTA=ethylene glycol-bis(2-aminoethyl ether) N,N,N′,N′, tetra-aceticacid

TES=N-tris (hydroxymethyl) methyl-2-aminoethane-sulfonic acid

HEPES=N-(2-hydroxyl ethyl) piperarine-N′-(2-ethanesulfonic acid)

PBS=phosphate buffered saline; HSA=human serum albumin

BSA=bovine serum albumin; ACD=citric acid, citrate, and dextrose

MPCD=methyl-β-cyclodextrin

EXAMPLE I

p26 was purified from encysted embryos of A franciscana (San FranciscoBay) purchased from San Francisco Bay Brand, Newark, Calif., USA.Purification steps were performed at 4° C. or on ice. Dried embryos (50g), were hydrated at 4° C. for 16 hours in sea water; filtered; washedwith cold 40 mM HEPES-KOH, pH 7.5, at 4° C., 70 mM NaCl, and 1 mM EDTA(buffer A); and homogenized in the same buffer with a Retsch motorizedmortar and pestle (Brinkman Instruments, Canada). The homogenate wascentrifuged (4000×g, 20 minutes) and the supernatant filtered through 6layers of cheesecloth, centrifuged again at 16 000×g for 40 minutes, andthen at 23 500×g for 30 minutes. Solid (NH₄)₂SO₄ was added to 40%saturation in the final supernatant. Precipitated proteins werecollected at 10 000×g for 30 minutes; dissolved in 20 mM Tris-HCl, pH8.15, 150 mM NaCl, 1 mM MgCl₂, and 0.1 mM EDTA (buffer B); and dialyzedovernight against this buffer. After dialysis, the solution was passedthrough a 0.45-mm filter, applied to a Source 15 Q ion-exchange column(Amersham Pharmacia Biotech), equilibrated, and developed at 2 mL/min inbuffer B. The column was washed with buffer B for 30 minutes, and alinear NaCl gradient (150-500 mM) was used for elution of p26 between235-270 mM NaCl. Fractions containing p26 were pooled; concentratedusing Centriprep-30 (Amicon); dialyzed against 40 mM HEPES-KOH, pH 7.5(buffer C), and 3.00 mM NaCl; further purified by gel filtration using aTSK-Gel G4000SW_(XL) column (0.78×30 cm, Toso Haas, Japan);equilibrated; and developed at 0.5 mL/min in buffer C and 300 mM NaCl.p26 was eluted between 9.5-10.5 mL of the buffer volume, and theresulting protein was more than 95% pure. The protein was concentratedto approximately 1 mg/mL with Centriprep-30; dialyzed against 50 mMTris-HCl, pH 8, and 2 mM EDTA (TE buffer); and centrifuged at 10 000×gfor 15 minutes. Further concentration and storage in buffer C led tounwanted insoluble aggregates. Aliquots were quick frozen in liquidnitrogen and stored at −70° C. Fractions from each step of purificationwere checked by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and/or Western immunoblotting with polyclonal antibodyto p26 and then pooled according to purity.

EXAMPLE II

293 cells and T293 cells were grown in T-25 flasks to ˜90% confluence.The cells were harvested by trypsinization according to standardprotocols. Briefly, the medium was removed from the cultures and theywere washed one time with 5 mL DPBS. Trypsin (1 mL of 0.05% in 0.53 mMEDTA-4Na) was added to the culture for ˜1 min and the flasks were rappedto dislodge the cells. Medium (4 mL) was added to stop the reaction, andthe cells were pelleted by centrifugation at 176×g for 5 min. The pelletwas suspended in 5-10 mL DPBS and the centrifugation step was repeated.The cell pellet was then suspended in air drying buffer lackingtrehalose (10 mM Hepes, 5 mM KCl, 65 mM NaCl, and 5.7% BSA with pH 7.2)at 1.4 million cells per mL. Aliquots (1.0 mL) were placed in 35 mmpolysterene Petri dishes and air-dried in a ThermoForma biosafetycabinet in specific marked locations in the center of the hood over 0-24hours. At various time points during drying, samples were removed forviability and water content analyses. Water contents were measuredgravimetrically in triplicate. For viability measurements, samples wererehydrated with 1 mL medium. 50 μL of cellular suspension was mixed with50 μL trypan blue and incubated at room temperature for 3 min. Cellswere visualized at 10× by light microscopy on a hemacytometer andcounted using five counts of 50-100 cells per 1 mm² hemocytometer gridsquare for each sample. Percent viability was calculated as the numberof cells excluding the dye divided by the total number of cells.Viability was plotted as a function of water content as themeans+/−standard deviation (for both variables), with the resultsillustrated in FIG. 1.

EXAMPLE III

293 cells and T293 cells were grown in T-25 flasks to ˜90% confluence.The cells were harvested by trypsinization according to standardprotocols. Briefly, the medium was removed from the cultures and theywere washed one time with 5 mL DPBS. Trypsin (1 mL of 0.05% in 0.53 mMEDTA-4Na) was added to the culture for ˜1 min and the flasks were rappedto dislodge the cells. Medium (4 mL) was added to stop the reaction, andthe cells were pelleted by centrifugation at 176×g for 5 min. The pelletwas suspended in 5-10 mL DPBS and the centrifugation step was repeated.The cell pellet was then suspended in air drying buffer containingtrehalose (10 mM Hepes, 5 mM KCl, 65 mM NaCl, 150 mM Trehalose, and 5.7%BSA with pH 7.2) at 1.4 million cells per mL. Aliquots (1.0 mL) wereplaced in 35 mm polysterene Petri dishes and air-dried in a ThermoFormabiosafety cabinet in specific marked locations in the center of the hoodover 0-24 hours. At various time points during drying, samples wereremoved for viability and water content analyses. Water contents weremeasured gravimetrically in triplicate. For viability measurements,samples were rehydrated with 1 mL medium. 50 μL of cellular suspensionwas mixed with 50 μL trypan blue and incubated at room temperature for 3min. Cells were visualized at 10× by light microscopy on a hemacytometerand counted using five counts of 50-100 cells per 1 mm² hemocytometergrid square for each sample. Percent viability was calculated as thenumber of cells excluding the dye divided by the total number of cells.Viability was plotted as a function of water content as themeans+/−standard deviation (for both variables), the results shown inFIG. 2.

EXAMPLE IV

293 cells and T293 cells were grown in T-25 flasks to ˜90% confluence.The cells were incubated in medium with 100 mM trehalose for 24 hours at37° C. to induce endocytotic loading. The cells were then harvested bytrypsinization according to standard protocols. Briefly, the medium wasremoved from the cultures and they were washed one time with 5 mL DPBS.Trypsin (1 mL of 0.05% in 0.53 mM EDTA-4Na) was added to the culture for˜1 min and the flasks were rapped to dislodge the cells. Medium (4 mL)was added to stop the reaction, and the cells were pelleted bycentrifugation at 176×g for 5 min. The pellet was suspended in 5-10 mL,DPBS and the centrifugation step was repeated. The cell pellet was thensuspended in air drying buffer containing trehalose (10 mM Hepes, 5 mMKCl, 65 mM NaCl, 150 mM Trehalose, and 5.7% BSA with pH 7.2) at 1.4million cells per mL. Aliquots (1.0 mL) were placed in 35 mm polysterenePetri dishes and air-dried in a ThermoForma biosafety cabinet inspecific marked locations in the center of the hood over 0-24 hours. Atvarious time points during drying, samples were removed for viabilityand water content analyses. Water contents were measured gravimetricallyin triplicate. For viability measurements, samples were rehydrated with1 mL medium. 50 μL of cellular suspension was mixed with 50 μL trypanblue and incubated at room temperature for 3 min. Cells were visualizedat 10× by light microscopy on a hemacytometer and counted using fivecounts of 50-100 cells per 1 mm² hemocytometer grid square for eachsample. Percent viability was calculated as the number of cellsexcluding the dye divided by the total number of cells. Viability wasplotted as a function of water content as the means+/−standard deviation(for both variables), with the results shown in FIG. 3.

EXAMPLE V

293 cells and T293 cells were grown in T-25 flasks to ˜90% confluence.The cells were incubated in medium with 100 mM trehalose for 24 hours at37° C. to induce endocytotic loading. The cells were then harvested bytrypsinization according to standard protocols. Briefly, the medium wasremoved from the cultures and they were washed one time with 5 mL DPBS.Trypsin (1 mL of 0.05% in 0.53 mM EDTA-4Na) was added to the culture for˜1 min and the flasks were rapped to dislodge the cells. Medium (4 mL)was added to stop the reaction, hand the cells were pelleted bycentrifugation at 176×g for 5 min. The pellet was suspended in 5-10 mLDPBS and the centrifugation step was repeated. The cell pellet was thensuspended in air drying buffer containing trehalose(10 mM Hepes, 5 mMKCl, 65 mM NaCl, 150 mM Trehalose, and 5.7% BSA with pH 7.2) at 1.4million cells per mL. Aliquots (1.0 mL) were placed in 35 mm polysterenePetri dishes and air-dried in a ThermoForma biosafety cabinet inspecific marked locations in the center of the hood. Samples were thenremoved and rehydrated with 1 mL medium. Following viability testing bytrypan blue exclusion (which used 50 mL), the remaining 950 mL wascombined with 7 mL medium, and replated in a T-25 flask. Parallelsamples were assayed for residual water content by gravimetric analysis.The cultures were incubated at 37° C., 90% relative humidity, and 5% CO₂for 24 hours, after which time the medium was removed and replaced withfresh medium. After incubation for 6 more days under the sameconditions, the medium was removed and the colonies were stained withHema 3 and counted. The results are illustrated in FIG. 4, which is agraph of the number of colonies formed after rehydration vs watercontent after drying the transfected 293 cells (T-293 cells) and thecontrol 293 cells (293-cells) to 0.3 gm. water/gm. dry weight and to 0.2gm. water/gm. dry weight, with both the transfected 293 cells and thecontrol 293 cells having trehalose internally and with the drying bufferfor both the transfected 293 cells and the control 293 cells having 150mM trehalose.

EXAMPLE VI

293 cells transfected to produce the protein P26 from Artemia wereloaded with trehalose for 24 hours by incubation at 37° C. in medium+100mM trehalose, which resulted in internally trehalose concentration inthe range 20-40 mM. The cells were dried by either air-drying orvacuum-drying and the viability following rehydration was compared bytrypan blue exclusion. The air-dried samples (50 μL) were placed at roomtemperature (˜20° C.) in a modified desiccator flushed with dry air atapproximately 200 mL/min. The vacuum-dried samples (50 pL) were placedin a vacuum chamber at room temperature and subjected to a vacuum ofapproximately 23 inches Hg. The residual water contents were measured bygravimetric analysis. The vacuum-dried samples show a significantly 1left-shifted curve as compared to the air-dried samples, indicating amuch higher viability at the lowest water contents.

EXAMPLE VII

Transfected 293 cells were dried in different physical configurations todetermine the effect of the physical structure of the sample onviability following rehydration. 293 cells transfected to produce theprotein P26 from Artemia were loaded with trehalose for 24 hours byincubation at 37° C. in medium+100 mM trehalose, which results in aninternal trehalose concentration in the range of 20-40 mM. The cellswere dried by either air-drying or vacuum-drying and the viabilityfollowing rehydration was compared by trypan blue exclusion. The airdried samples (50 μL) were placed at room temperature (˜20° C.) in amodified desiccator flushed with dry air at approximately 200 mL/min.The vacuum-dried samples (50 μL) were placed in a vacuum chamber at roomtemperature and subjected to a vacuum of approximately 3 inches Hg. Theresidual water contents were measured by gravimetric analysis. Cellswere air-dried or vacuum-dried from either a 50 μL thin film or a 50 μLrounded droplet. Above 2 gH₂O/g dry weight, there is little effect ofthe physical structure. But, at the lowest water contents, theviabilities are 20-40% higher when the samples are dried in a roundeddroplet instead of a thin film.

EXAMPLE VIII

Average viabilities (+/−SD) of vacuum-dried 293 cells and T293 cellswhen dried as rounded 50 μL droplets (beads). 293 cells transfected toproduce the protein p26 from Artemia and control 293 cells were loadedwith trehalose for 24 hours by incubation at 37° C. in medium+100 mMtrehalose, which results in an internal trehalose concentration in therange of 20-40 mM. The cells were dried by vacuum-drying and theviability following rehydration was compared by trypan blue exclusion.The vacuum-dried samples (50 μL) were placed in a vacuum chamber at roomtemperature and subjected to a vacuum of approximately 3 inches Hg. Theresidual water contents were measured by gravimetric analysis. Althoughboth types of cells show improved viability with this combination, ascompared to air-dried samples, the transfected cells still show highersurvival than the standard 293 cells at water contents below 2gH₂O/g dryweight. Using this combination of treatments, the viability for thetransfected cells is approaching 40% at 0.2gH₂O/g dry weight. This is asignificant improvement over methods described in previous disclosures.

CONCLUSION

Embodiments of the present invention provide that mammalian cells (e.g.,T-293 cells) transfected with the stress protein p26 and loaded withtrehalose, a sugar found at high concentrations in organisms thatnormally survive dehydration, survived drying at water contents of about0.5 gm water/gm dry weight cells, and below about 0.5 gm water/gm dryweight cells. Drying of the cells may be in any suitable manner, such asair drying or vacuum drying. Control mammalian cells not transfectedwith the stress protein p26, by contrast to the transfected mammaliancells, have diminished survival at water contents as high as 2 gmwater/gm dry weight cells. Thus, transfection of mammalian cells withp26 and loading with trehalose improves the ability to dry mammaliancells, particularly mammalian nucleated cells.

EXAMPLE IX

This Example sets forth a procedure for conducting an exemplar assay forascertaining entry of a solute into a cell.

Loading of Lucifer Yellow CH into Cells. A fluorescent dye, luciferyellow CH (LYCH), can be used as a marker for penetration of cellmembranes by a solute. Washed cells are incubated in the presence of1-20 mg/ml LYCH. Incubation temperatures and incubation times can bechosen as desired. After incubation, the cells are spun at 20× at 14,000RPM on a table centrifuge, resuspended in buffer, spun down for 20 s inbuffer and resuspended. Cell counts are obtained on a suitable counter,such as a hemacytometer, and the samples pelleted (for example, bycentrifugation for 45 s 25 at 14,000 RPM, table centrifuge). The pelletis lysed in 0.1% Triton buffer (10 mM TES, 50 mM KCl, pH 6.8). Thefluorescence of the lysate is measured on a Perkin-Elmer LSSspectrofluorimeter with excitation at 428 nm (SW 10 nm) and emission at530 run (SW 10 nm). Uptake is calculated for each sample as nanograms ofLYCH per cell using a standard curve of LYCH in lysate buffer.

Visualization of cell-associated Lucifer Yellow. LYCH loaded plateletscan be viewed on a fluorescence microscope (Zeiss) employing afluorescein filter set for fluorescence microscopy. Cells can be studiedeither directly after incubation or after fixation with 1%paraformaldehyde in buffer. Fixed cells can be settled on poly-L-lysinecoated cover slides and mounted in glycerol.

Quantification of Trehalose and LYCH Concentration. Uptake is calculatedfor each sample as micrograms of trehalose or LYCH per cell. Theinternal trehalose concentration can be calculated assuming a standardcell radius and by assuming that 50% of the cell volume is taken up bythe cytosol (rest is membranes). The loading efficiency was determinedfrom the cytosolic trehalose or LYCH concentration and the concentrationin the loading buffer.

EXAMPLE X

This Example sets forth materials and methods for studies of the effectof arbutin in the drying and rehydration of MSCs.

Materials and Methods

Materials

Tissue culture reagents were from Invitrogen (Carlsbad, Calif.), unlessotherwise stated. Tissue culture disposables were from Nalge NuncInternational (Rochester, N.Y.). Trehalose was from Cargill.(Minneapolis, Minn.). Equipment for western blots, and reagents werefrom Bio-Rad (Hercules, Calif.) unless otherwise stated. Bovine serumalbumin (BSA), and glycine was from Research Organics (Cleveland, Ohio),Bromodeoxyuridine, and anti-bromodeoxyuridine, (mouse IgG_(l),monoclonal PRB-1, Alexa Fluor® 488 conjugate) was obtained fromMolecular Probes (Eugene, Oreg.). Arbutin, ascorbic acid, silvernitrate, propidium iodide, and dexamethasone were from Sigma-Aldrich (StLouis, Mo.) and β-glycerophosphate was from Calbiochem (San Diego,Calif.).

Cell Culture

Human MSCs previously isolated from bone marrow and expanded in vitro topassage number 1 were a gift from Osiris Therapeutics (Baltimore, Md.)and were shipped to UC Davis in liquid nitrogen. The cells were grown inDulbecco's modified Eagle medium-low glucose, with 10% FBS (Hyclone,Logan, Utah) at 37° C. with 5% CO₂ and 90% RH. The cells were used upthrough passage number 4 at a level of 90-95% confluence. Cells wereharvested by washing once with Dulbecco's PBS (DPBS) and incubating for5-7 min with trypsin-EDTA [0.05% trypsin, and 0.53 mM EDTA-4Na]. Thiscell suspension was pelleted at 167×g for 10 min and resuspended inmedium or the specified buffer. Unused cells were counted beforefreezing and were frozen in mixture of 10% DMSO, 5% human serum albumin,and 70% Plasma Lyte A (both from Baxter Healthcare Corp., Deerfield,Ill.) until they were needed.

Solute Loading

Cells were grown in T-75 flasks to 90-95% confluence and loaded withextracellular solutes. Briefly, medium was removed from the flasks andreplaced with MSC growth medium containing 100 mM trehalose or 70 mMtrehalose plus 40 mM arbutin for 24 hours at 37° C. Followingincubation, the cells were washed once with 10 mL DPBS and harvested bytrypsinization, as described above. The MSCs were then transferred toone of three different drying buffers. The control (no trehalose) dryingbuffer contained 10 mM Hepes, 5 mM KCl, 140 mM NaCl, with pH 7.2. Thetrehalose-only drying buffer contained 10 mM Hepes, 5 mM KCl, 65 mMNaCl, 150 mM trehalose, and 5.7% BSA with pH 7.2, and thetrehalose-plus-arbutin drying buffer included 10 mM Hepes, 5 mM KCl, 30mM NaCl, 150 mM trehalose, 70 mM arbutin, and 5.7% BSA with pH 7.2.

Vacuum-Drying

The samples were dried in 50 μL aliquots in the shape of roundeddroplets in the caps of Eppendorf microfuge tubes (Online Products,Petaluma, Calif.), at room temperature under a vacuum (pressure ˜3 inHg). At various time points, parallel samples were removed forassessment of viability and residual water content.

Viability was measured by propidium iodide (PI) exclusion as follows.Rehydrated MSCs were incubated in 2 μg/ml PI for 10 min, then loaded ona hemacytometer (Hausser Scientific, Horsham, Pa.) and examined using anOlympus BX 61 fluorescence microscope (Miami, Fla.). The total number ofcells was counted by differential interference contrast microscopy, andthe number of dead cells was counted by fluorescence using the Tritc/Di(U-N41002a) from Chroma Technology Corporation (Rockingham, Vt.). Fivecounts of 50-100 cells per 1 mm² hemocytometer grid square were takenfor each sample.

Gravimetric analysis was used to measure the residual water content ofthe vacuum-dried samples. Samples were weighed on a R 180 D, modelSartorius balance (Westbury, N.Y.) immediately following removal fromthe vacuum chamber (=vacuum-dried sample) and again following completewater removal by incubation at 60° C. under vacuum (pressure˜3 in Hg)for 24-48 h until a constant mass was achieved (=anhydrous sample). Thedifference between the anhydrous weight (includes vessel) and the vesseltare was taken as the dry weight of the sample. The difference betweenthe weight of the vacuum-dried sample and that of the anhydrous sample(both include vessel) was taken as the water weight. The residual watercontents are reported as the g water per g dry weight of the sample (gH₂O/g dry weight).

Rehydration and Cellular Recovery

Vacuum-dried samples were rehydrated with excess medium (150 uL persample) and mixed by gentle pipetting. The rehydrated cells were thenreplated in Lab-Tek slides and incubated overnight. They were thenincubated in medium containing 10% alamarBlue (Bio Source, Camarillo,Calif.) for 24 hours. AlamarBlue is reduced by actively metabolizingcells, and only the reduced from is fluorescent. Thus, cellularmetabolism can be monitored by alamarBlue fluorescence (REF). Aliquotsof medium (1.0 mL) were measured on a Perkin Elmer LS50B luminescencespectrometer (Ex 530 nm, Em 585 nm).

Cell division was monitored by incorporation of bromodeoxyuridine(BrdU). Rehydrated cells were replated as described above and culturedfor three weeks in order to gain enough cells to acquire accurate cellcounts. The cells were then re-plated at a 1:3 split and pulsed for 2 dwith 10 μM BrdU. The samples were washed twice with 1 mL DPBS, and fixedby incubation with 1:1 of DPBS: methanol overnight at 4° C. temperature.The cells were permeabilized with 0.01% Tritron X 100 solution in DPBSfor 5 minutes, washed and incubated with 2N HCl for 30 minutes at 37° C.They were then blocked with 1% BSA for 45 minutes at 37° C. and werestained with fluorescently tagged antibodies to the BrdU (Alexa 488)(1:20 dilution) for 45 minutes at 37° C. and propidium iodide (2 μg/mLPI for 10 min). Using an Olympus BX 61 fluorescence microscope, thetotal cellular population was visualized by the propidium iodide (red)staining, and dividing cells were visualized with the Alexa 488 (green)staining using appropriate TRITC and FITC channels, respectively.Statistical analysis for this and other experiments was conducted usinga one-way analysis of variance (ANOVA) with Sigma-Stat software (JandelScientific, San Rafael, Calif.).

Osteogenic Differentiation

MSCs were dried and rehydrated as described above. The cells were thenreplated in Lab-Tek slides and incubated under normal growth conditions(37° C., 5% CO₂, 90% RH), either in the presence of absence ofosteogenic supplements (OS). OS medium consisted of D-MEM supplementedwith 10% FBS (v/v), 0.1 μM dexamethasone, 50 μM ascorbicacid-2-phosphate, and 10 mM β-glycerophosphate. The cells were fed every3-4 days by removing and replacing the medium (+/−osteogenicsupplements). The cells were grown for two weeks (+/−OS supplements)before usage in the calcium deposition assay.

Differentiation along the osteogenic lineage was assessed by conductinga von Kossa stain for calcium deposition. Briefly, one well of a 2-wellLab-Tek slide was used for each sample. The medium was removed and allwells were rinsed twice with DPBS, and then fixed with 10% formalin,followed by two additional washes with DPBS. To each well, 1 mL 2% AgNO₃was added, and the plates were incubated in the dark for 10 min.Following the AgNO₃ incubation, all wells were rinsed three times withwater, leaving the last rinse on the cells. The plates were placed on awhite background with the lids removed and exposed to bright light for15 min. Finally, the wells were rinsed again thoroughly with water andair dried in the hood. Observation of a dark brown stain was taken asindication of calcium deposition.

To quantify the calcium deposition, triplicate samples of MSCs wereloaded and dried to various water contents with trehalose alone ortrehalose plus arbutin, as described above. The samples were rehydratedwith excess medium and cultured in the presence of osteogenicsupplements for two weeks. Medium was then removed from the samples,which were dissolved with 1 N HCl (1 mL). The calcium present wasmeasured using the calcium quantitation kit from Cima Scientific(Dallas, Tex.), by comparison to a standard curve according tomanufacturer's protocols. This assay is based on the principle ofo-cresolphthalein binding to calcium which forms a purple complex thatcan be measured spectrophotometrically at 650 nm.

Western Blot Analysis

MSCs were incubated for 24 h in medium containing 10, 25, 50, or 100 mMarbutin at 37° C., or were not incubated with arbutin (controls). Thecells were collected by trypsinization, washed with DPBS, counted on ahemacytometer, and transferred into triple detergent lysis buffer (50 mMTris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN₃, 0.1% SDS, 5 mM pefablock, 1μg/mL aprotinin, 1% nonidet P-40, and 0.5% sodium deoxycholate) for 30min with ˜5 sec vortex intervals every ˜5 min. The suspensions werepelleted on an Eppendorf microfuge at 15,000 rpm for 15 min at 4° C.,and the supernatants were recovered. The cell lysates were analyzed forprotein content by the Lowry method (BioRad QC protein assay kit), anddiluted 1:1 into 2× loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20%glycerol, 10% beta mercaptoethanol, and bromophenol blue). The proteinswere analyzed by SDS PAGE, using a 13% gel and 20 μg protein per laneand transferred onto PDVF membranes in Towbin buffer (25 mM Tris base,192 mM glycine, 20% methanol, pH 8.3). The blot was cut in half, sostaining for HSP70 and HSP27 could be accomplished simultaneously. Theblots were blocked with 5% non-fat dry milk, and stained with mouseanti-HSP70 (SPA 810, 1:1000 dilution), or mouse anti-HSP27 (SPA 800,1:1000 dilution), both from Stressgen Biotechnologies Corporation(Victoria, B.C, Canada), then stained with goat anti-mouse antibodyconjugated to alkaline phosphatase, and visualized by incubating withNBT/BCIB (both from Pierce Biotechnology, Inc, Rockford, Ill.). Theblots were scanned and quantified using the program Quantity One fromBio-Rad.

EXAMPLE XI

This Example reports the results of the effects of arbutin on survivalof MSCs during drying and rehydration.

Mesenchymal Stem Cells Survive Drying to 0.3 g H₂O/g Dry Weight

MSCs were loaded with trehalose by a 24-h incubation in growth mediumcontaining 100 mM trehalose. The trehalose-loaded cells were harvestedby trypsinization and transferred into drying buffer containingtrehalose (10 mM Hepes, 5 mM KCl, 65 mM NaCl, 150 mM trehalose, and 5.7%BSA with pH 7.2). In parallel, control cells were harvested bytrypsinization, and transferred into drying buffer lacking trehalose (10mM Hepes, 5 mM KCl, 140 mM NaCl, pH 7.2). Aliquots of cell suspension(50 μL at 1.25×10⁶ cells/mL) were dried in the caps of Eppendorfmicrofuge tubes by exposure to vacuum (pressure ˜6 in Hg) for a periodof 0-5 h. Viability decreased with residual water content. Theprotective role of trehalose was clear from the data, especially below2.0 g H₂O/g dry weight, thus trehalose was included in all thesubsequent experiments. It was found that ˜50% of the cells surviveddrying to ˜0.3 g H₂O/g dry weight. That is, to our knowledge, thehighest viability reported for nucleated cells taken to this level ofdehydration.

Arbutin was also tested as a possible protectant for the MSCs duringdrying. MSCs were loaded with the protective solutes by a 24-hincubation in growth medium containing 100 mM trehalose or 70 mMtrehalose and 40 mM arbutin at 37° C., 5% CO₂, and 90% RH. The sampleswere then harvested by trypsinization and transferred into drying buffercontaining trehalose only (10 mM Hepes, 5 mM KCl, 65 mM NaCl, 150 mMtrehalose, and 5.7% BSA with pH 7.2) or trehalose plus arbutin (10 mMHepes, 5 mM KCl, 30 mM NaCl, 150 mM trehalose, 70 mM arbutin, and 5.7%BSA with pH 7.2). The samples were vacuum dried to four different watercontents spanning a large range for residual water (1.34-0.23 g H₂O/gdry weight) and rehydrated with excess medium. The viabilities weremeasured by propidium iodide (PI) exclusion.

Arbutin provided neither a benefit nor liability to the samplesimmediately following rehydration, as the trehalose-only andtrehalose-plus-arbutin samples showed no significant difference insurvival for any water content tested (P=0.174). This result contrastedwith the effect of other antioxidants, such as epigallocatechin gallate(EGCG), glutathione, or glutathione ester, which all caused largedecreases in viability measured immediately after rehydration. This ledus to hypothesize that arbutin might be a valuable protective compoundfor the MSCs, since there was no drop in immediate viability, and thebeneficial effects of arbutin were likely to appear over time in therehydrated samples.

Arbutin Enhances Recovery of Vacuum-dried MSCs

One method for measuring the recovery of the rehydrated cells is toexamine the cellular metabolism of the rehydrated samples. The dyealamarBlue is reduced by actively metabolizing cells, and only thereduced form is fluorescent. MSCs were loaded and vacuum-dried tovarious water contents with trehalose only or trehalose plus arbutin, asdescribed above. The samples were dried and rehydrated under sterileconditions, and then re-plated in medium containing 10% alamarBlue.After 24 h incubation, the fluorescence of the medium was measured (Ex530, Em 585) for all samples.

At the highest water contents tested (1.34 and 0.56 g H₂O/g dry weight),there was no difference in the ability of the rehydrated cells to reducealamarBlue (P=0.588). As the water content was reduced below 0.4 g H₂O/gdry weight, however, a significant difference appeared between thearbutin containing samples and the controls (P=0.030). In fact, at 0.38g H₂O/g dry weight, there was almost a four-fold increase in thefluorescence of the alamarBlue from the arbutin containing samples, ascompared to the trehalose-only samples. In both cases, the fluorescencedropped at the lowest water contents, but in the arbutin containingsamples this decrease occured at a much lower water content (0.23 gH₂O/g dry weight) than in the control samples (0.38 g H₂O/g dry weight).This result indicates that at water contents below 0.5 g H₂O/g dryweight, arbutin can provide a significant advantage to cell health overtime following rehydration.

Another, more stringent, test for rehydrated MSCs is whether the cellscan grow and divide following rehydration. Bromodeoxyuridine (BrdU) canserve to label cells that are actively dividing, as it is onlyincorporated into newly synthesized DNA. MSCs were vacuum-dried in thepresence or absence of arbutin, as described above. The cells wererehydrated under sterile conditions, re-plated, and cultured for 3weeks, after which they were split 1:3 and pulsed for 2 days with 10 μMBrdU. The cultures were then washed, permeabilized, and stained withfluorescently tagged antibodies to the BrdU (Alexa 488) and propidiumiodide. The total cellular population was visualized by the propidiumiodide staining, and the dividing cells were visualized with thefluorescent antibodies to BrdU (nuclei stained green).

The arbutin-containing samples had significantly higher cell counts atall except the highest water content tested (P=0.001). This differencewas particularly dramatic at the two lowest water contents tested (0.33and 0.27 g H₂O/g dry weight). In fact, at 0.27 g/g, no cells remained inthe trehalose-only samples, but a considerable number of cells werestill present in the arbutin containing samples.

Of the cell population present, the line plots show that approximately70-80% were capable of cell division. There was very little differencebetween the samples dried in the presence or absence of arbutin for thisparameter (P=0.656). The finding that such a large percentage of therehydrated cells could incorporate BrdU into newly synthesized DNAsuggests excellent retention of normal physiological processes, and thelarger cell counts found in samples dried in the presence of arbutinprovides further evidence that this hydroquinone enables enhancedrecovery of dried and rehydrated MSCs.

Arbutin Enhances Osteogenic Differentiation of Vacuum-dried MSCs

We further investigated whether the rehydrated MSCs are capable ofdifferentiation down the osteogenic pathway, as an indication of whetherstem cells would retain differentiation capabilities followingdehydration and storage. Cells were loaded and vacuum-dried to 0.37 gH₂O/g dry weight in the presence of trehalose or trehalose plus arbutin.The samples were then rehydrated under sterile conditions and culturedfor two weeks in the presence or absence of osteogenic supplements (OS,0.1 μM dexamethasone, 50 μM ascorbic acid-2-phosphate, and 10 mMβ-glycerophosphate). Differentiation along the osteogenic lineage wasassessed by conducting a von Kossa stain for calcium deposition.Observation of a dark brown stain following treatment with AgNO₃ wastaken as indication of calcium deposition.

The samples grown in the absence of osteogenic supplements were negativefor von Kossa staining, as expected. Both samples treated with theosteogenic supplements were positive for von Kossa staining, but it wasmuch more pronounced in the samples that were loaded and dried witharbutin and trehalose, in comparison to samples that were loaded anddried with trehalose alone. These results show that the dried andrehydrated cells are indeed competent to differentiate along one of thenormal developmental pathways, and that arbutin augments this ability.

Since the difference in the von Kossa staining was quite strikingbetween the samples dried in the presence and absence of arbutin, wequantified the calcium deposition using o-cresolphthalein binding tocalcium, which forms a purple complex that can be measuredspectrophotometrically. Triplicate samples of MSCs were loaded and driedto various water contents with trehalose alone or trehalose plusarbutin. The samples were rehydrated under sterile conditions andcultured in the presence of osteogenic supplements for two weeks, asdescribed above. The samples were then dissolved with 1 N HCl and thecalcium measured using a kit from Cima Scientific and comparison to astandard curve.

The results show that, similar to other measurements of cell health,there was little difference between the samples dried to the highestwater contents. In samples dried to 0.47 g H₂O/g dry weight, calciumdeposition found in MSC samples dried in the presence or absence ofarbutin was virtually identical. In contrast, as water was removed, thedifference between the samples became dramatic. At a water content of0.30 g H₂O/g dry weight, arbutin caused a 25-fold increase in theability of the cells to deposit calcium. Clearly, arbutin conferred adistinct advantage to the dried MSCs, an advantage which appears overtime after rehydration and enables the samples to more effectivelydifferentiate when conditions are appropriate.

Arbutin Induces Expression of HSP70 in MSCs

The chemical structure for arbutin resembles that of aspirin andsalicylic acid, two known inducers of the heat shock response. We,therefore, addressed the hypothesis that one mechanism by which arbutincould impart some protective effect under stressful conditions isthrough inducing the expression of heat shock proteins in the MSCs. AWestern blot analysis was conducted for HSP70 and HSP27 on samplesincubated for 24 h at 37° C. in the presence of increasingconcentrations of arbutin.

Both the blots themselves and a line plot showing the quantitation ofthe immunostained protein bands indicate that arbutin causes adose-dependent increase in the expression of HSP70, but not HSP27. Theeffect on HSP70 at 50 mM arbutin (which is similar to the concentrationduring the loading phase in the drying experiments), although not asdramatic as that at 100 mM, is still significant (P=0.002 by ANOVA test,SigmaStat software). This result is consistent with a protective effectof arbutin through induction of endogenous heat shock proteins. However,arbutin also has several other beneficial properties, as mentionedabove. Thus, it is quite likely that the full protective role of arbutinis the result of a complex series of effects at both the membrane andcellular levels.

EXAMPLE XII

This Example discusses the results set forth in the previous Example.

The current study has addressed the critical issue of whether dried andrehydrated mesenchymal stem cells can function normally in response todifferentiation signals. Two protective compounds were investigated,trehalose and arbutin. Cells dried in the absence of both solutes didnot survive well below 2.0 g H₂O/g dry weight. This suggests thatearlier reports, in which water contents were not quantified, but thatshowed high cellular viability or attachment to the growth surface,after drying in the absence of trehalose (e.g. Gordon et al., 2001),were actually investigations of samples containing relatively high watercontents.

Samples loaded and dried in the presence of trehalose and arbutin showedlittle difference in viability from samples loaded and dried withtrehalose alone when measured immediately following rehydration. Thisapparently negative result was actually a promising indication thatarbutin could serve as a useful tool in preserving the MSCs duringdehydration. Antioxidants can help protect against damaging reactiveoxygen species (ROS) generated under mostly dehydrated conditions, butother antioxidants tested, such as EGCG and glutathione, caused largedecreases in survival. Thus, the finding that arbutin was not disruptiveto membrane integrity immediately after rehydration led to thepossibility of exploiting its many protective properties during thevarious stages of loading, drying, rehydration, and culturing of theMSCs.

Arbutin does not have a universally protective effect, however. In fact,the striking benefit of arbutin to dried and rehydrated MSCs was celltype-specific, as arbutin was toxic to 293H cells. It is wellestablished that the role of arbutin as either stabilizer ordestabilizer depends on the lipid composition of the membranes present,and this can help to explain the contrasting effects on different celltypes.

Besides arbutin, other amphiphilic compounds are common in desiccationtolerant plant tissues, such as seeds and pollen grains. These compoundsquite often partition into membranes to a greater extent during dryingthan in the fully hydrated state, which is likely to be the case forarbutin as well. The protective role of these compounds remainssomething of a puzzle, as they often cause membrane leakage when testedin vitro. Their presence in tissues and organisms capable ofwithstanding dehydration must indicate that their beneficial effects(most are strong antioxidants) outweigh their damaging properties, atleast in the region of the specific target membranes where they arefound. The current findings indicate that, under specialized conditions,amphiphiles such as arbutin can be used in the preservation of cells ortissues unrelated to those from which they came.

In the days and weeks following rehydration, the samples dried in thepresence of arbutin showed much stronger recovery, as measured bycellular metabolism and cell count. In contrast, there was nosignificant difference between the samples dried in the presence orabsence of arbutin with regard to BrdU incorporation. This was verylikely a result of experimental protocol, however. In order to haveenough cells for an accurate count, the rehydrated cells had to becultured for three weeks before the BrdU pulsing could take place.During this time, the unhealthy cells could have been lost due to suchthings as poor attachment. This could have led to the high percentages(70-80%) of the cell populations that were capable of division.Nevertheless, the finding that both treatments produced cell populationscapable of cell division, as measured by BrdU incorporation, is asignificant advancement in the effort to preserve nucleated cells.Further, arbutin-treated samples did show a strong advantage in relationto the number of cells present after three weeks, especially when thesamples were taken to water contents below 0.5 g H₂O/g dry weight. Incombination with the results on osteogenic differentiation, these dataconfirm the ability of arbutin to aid recovery of the dried andrehydrated MSCs.

Based on the similarity of the chemical structure of arbutin with knowninducers of the heat shock response, a Western blot analysis for HSP70and HSP27 was conducted. Indeed, arbutin caused a dose dependentincrease in the expression of HSP70. Arbutin has been shown to increasethe fluidity of dry and hydrated model membranes, because it insertswith its phenol moiety into the bilayer, and lowers the gel to liquidcrystalline phase transition temperature. This is similar to the effectof other fluidizing agents, such as benzyl alcohol, which are known tolower the temperature at which the heat shock response is activated.Thus, arbutin's effect of inducing the expression of HSPs correlateswell with the membrane trigger hypothesis for induction of the heatshock response.

It is less likely that arbutin induced the expression of heat shockproteins in the MSCs by causing osmotic stress. Osmotic stress can causesuch a response, but the concentrations necessary are much higher. Thehighest concentration of arbutin used (100 mM) is not sufficient tocause expression of HSPs by this mechanism. Further, 100 mM trehalosehad the opposite effect, and actually decreased the expression of heatshock proteins in the MSCs. We therefore suggest that trehalose mightlower the level of stress “perceived” by the cells and thus inhibit theheat shock response to a certain degree, a hypothesis we are currentlyexploring.

The increased expression of heat shock proteins could serve to stabilizeproteins and membranes in the MSCs under stressful conditions. Inaddition, HSP70 has been shown in inhibit apoptosis caused by theleakage of cytochrome C from the mitochondria. Thus, the induction ofthese proteins could be one main mechanism by which arbutin aidsrecovery of the MSCs following rehydration.

It is likely that inducing the heat shock response is not the onlymechanism by which arbutin affects the cells, however. Arbutin has manyvaried effects on membranes, including inserting into the lipid bilayerat the phenol moiety, decreasing the phase transition of dry lipid,preventing enzymatic lipid hydrolysis, acting as an anti-oxidant,relaxing negative curvature and stabilizing the lamellar phase ofmembranes containing non bilayer-forming lipids, and stabilizing certaincompositions of membranes to freeze-thaw and drying stresses. Thus, itis reasonable that the added protection that arbutin affords to the MSCsduring drying is a complex process involving more than one pathway.

In summary, MSCs loaded and dried in the presence of trehalose showedalmost 60% viability after drying to 0.38 g H₂O/g dry weight. Includingarbutin in the loading and drying media did not change the viabilitywhen measured immediately after rehydration, but dramatically increasedrecovery of the MSCs, as measured by metabolism and cell count. Bothtreatments produced rehydrated cells capable of cell division, asmeasured by BrdU incorporation. When the cells were induced todifferentiate down the osteogenic lineage, both treatments resulted inpositive von Kossa staining. However, when the cells were dried to thelower water contents, arbutin caused nearly a 25-fold increase in thecells' ability to deposit calcium under osteogenic conditions. Theeffects of arbutin are likely to result from more than one mechanism,but one possible candidate is the induction of endogenous heat shockproteins, which was shown by Western blot analysis to be adose-dependent effect of arbutin in MSCs.

EXAMPLE XIII

This Example shows the transfection of cells with an examplar apoptosisinhibitor and expression of HSPs in cells.

Apoptosis was monitored during dry storage of CANARY cells. CANARY cellsare murine B cells designed for use in biosensors. See, Rider et al.,Science 301:213-215 (2003). Each line of CANARY cells is a clonespecific to detect a certain antigen (e.g. anthrax, plague, smallpox,etc). When the cells detect their specific antigen (or are exposed to anIgM), the internal calcium concentration increases. Because the cellsare engineered to express the jellyfish protein aqueorin, when theinternal calcium concentration increases, they give off a burst of lightthat is measurable by a bioluminometer. The presence of the reporterprotein makes them convenient to work with; however, the resultsobtained are expected to be generally applicable.

CANARY cells (4 flasks) were prepared for drying by a 24-h incubation ingrowth medium, in the presence or absence of 75 mM trehalose, in thepresence or absence of 30 μM OPH-109, a pan caspase apoptosis inhibitor(MP Biomedicals), in the presence or absence of 30 μM Caspase 1inhibitor II and in the presence or absence of 20 μg /ml Bcl -xL (acytochrome c release inhibitor). The cells were vacuum-dried in 50 μLdroplets under the same four conditions at 25° C., to a residual watercontent of 0.49 g H₂O/g dry weight. Samples were protected bytrehalose+OPH-109+Bcl -xL. Viability and apoptosis were quantified usingflow cytometry immediately following drying and rehydration, or after 24or 48 h storage in individually sealed vacuum packets at 4° C. in thedark. The cells were loaded and dried in the presence of both trehaloseand OPH-109, which produced nearly 70% viable cells and only ˜25%apoptotic cells following rehydration. We got same results in thepresence of trehalose and OPH and Caspase 1 inhibitor II or Bcl-xL, butthe effect of Bcl-xL was better than Caspase 1 inhibitor II on thestorage process and after storage for 24 h, the viability was stillgreater than 30%.

We also investigated the bioluminescence after drying CANARY B cells inthe presence or absence of 75 mM trehalose, in the presence of bothtrehalose+30 μM OPH-109, as described above, to a residual water contentof 0.71 g H₂O/g dry weight. The bioluminescence was quantified usingSirius Luminometer immediately following drying and rehydration, usingIgM as stimulator. The cells that were dried in the absence of trehaloseshowed a much lower signal in comparison to the undried controls thandid the cells loaded and dried in the presence of trehalose ortrehalose+OPH. This suggests that trehalose and OPH are very helpful forCANARY B cells during drying

The presence of heat shock proteins in CANARY B cells was investigatedby Western blot analysis and immunoostaining. The cells were washed withDPBS, counted on a hemacytometer, and transferred into triple detergentlysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3, 0.1% SDS,5 mM pefablock, 1 μg/mL aprotinin, 1% nonidet P-40, and 0.5% sodiumdeoxycholate) for 30 min with ˜5 sec vortex intervals every ˜5 min. Thesuspensions were pelleted on an Eppendorf microfuge at 15,000 rpm for 15min at 4° C., and the supernatants were recovered. The cell lysates wereanalyzed for protein content by the Lowry method (BioRad QC proteinassay kit), and diluted 1:1 into 2× loading buffer (125 mM Tris-HCl, pH6.8, 4% SDS, 20% glycerol, 10% beta mercaptoethanol, and bromophenolblue). The proteins were analyzed by SDS PAGE, using a 13% gel and 20 μgprotein per lane and transferred onto PDVF membranes in Towbin buffer(25 mM Tris base, 192 mM glycine, 20% methanol, pH 8.3). The membraneswere blocked with non-fat milk and probed with antibodies to HSP110,HSP90, HSP70, HSP60, HSP27, and α-B-crystallin, as well as secondaryantibodies conjugated to alkaline phosphatase. The CANARY B cellsstrongly expressed HSP110, HSP90, and HSP60.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for loading a disaccharide into mammalian nucleated cells,comprising: contacting said cells for at least 2 hours with a solutioncomprising at least one disaccharide, thereby loading the cells withdisaccharide to produce disaccharide-loaded mammalian nucleated cells.2. A method of claim 1, wherein said cells are selected from the groupconsisting of stem cells, immune system cells, and epithelial cells. 3.A method of claim 1, wherein said contacting is for 10 hours.
 4. Amethod of claim 1, wherein said contacting is for 24 hours.
 5. A methodof claim 1, wherein said disaccharide is trehalose.
 6. A method of claim1, wherein said solution further comprises not more than 3% dimethylsulfoxide.
 7. A method for increasing survival of mammalian nucleatedcells following drying and rehydration, comprising: (a) contacting saidcells with a solution comprising at least one disaccharide for at least2 hours, thereby producing disaccharide-loaded cells, (b) drying saiddisaccharide-loaded cells to a residual water content between 0.2 and0.5 gram water per gram of dry weight, and (c) rehydrating said cells,thereby increasing survival of the cells.
 8. A method of claim 7,wherein said contacting is for 24 hours.
 9. A method of claim 7, whereinsaid cells are selected from the group consisting of stem cells, immunesystem cells, and epithelial cells.
 10. A method of claim 7, whereinsaid disaccharide is trehalose.
 11. A method of claim 7, wherein saidcells further comprise a heat shock protein.
 12. A method of claim 11,wherein said heat shock protein is induced by exposing said cells to aheat shock.
 13. A method of claim 12, wherein said heat shock consistsof raising the temperature of medium contacting the cells to 42-44° C.for one hour, and then allowing the temperature of the medium to drop to36-38° C.
 14. A method of claim 11, wherein said heat shock protein isintroduced into the cells by contacting said cells with a solutioncomprising said protein.
 15. A method of claim 11, wherein said heatshock protein is expressed from a nucleic acid sequence introduced intosaid cells.
 16. A method of claim 11, wherein said heat shock protein isp26 from Artemia franciscana.
 17. A method of claim 7, further whereinsaid cells are contacted with a solution comprising an apoptosisinhibitor.
 18. A method of claim 17, wherein said apoptosis inhibitor isselected from the group consisting ofN-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone (inwhich the aspartyl residue is o-methylated or non-o-methylated), caspaseI inhibitor II, calpain inhibitor, and Bcl-xL.
 19. A method of claim 7,further wherein said cells are contacted by a solution comprisingarbutin or hydroquinone, provided that said cells are not 293 cells or Bcells.
 20. A method of claim 7, further wherein said cells are contactedby a solution comprising not more than 3% dimethyl sulfoxide.
 21. Amethod of claim 7, further wherein said cells are contacted by asolution comprising a heat shock protein and an apoptosis inhibitor. 22.A method of claim 21, wherein said solution further comprises not morethan 3% dimethyl sulfoxide.
 23. A method of claim 19, wherein said cellsare dried in a medium comprising arbutin or hydroquinone.
 24. A methodof claim 7, wherein said cells are dried in rounded droplets of dryingbuffer.
 25. A method for increasing survival of mammalian nucleatedcells following drying and rehydration, comprising: (a) contacting saidcells with a solution comprising an apoptosis inhibitor, thereby loadingthe cells with said apoptosis inhibitor, to produce apoptosisinhibitor-loaded cells, (b) drying said apoptosis inhibitor-loadedcells, and (c) rehydrating said cells, thereby increasing survival ofthe cells.
 26. A method of claim 25, wherein said apoptosis inhibitor isselected from the group consisting ofN-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone (inwhich the aspartyl residue is o-methylated or non-o-methylated), CaspaseI inhibitor II, Calpain inhibitor, and Bcl-xL.
 27. A method of claim 25,wherein said cells are selected from the group consisting of stem cells,immune system cells, and epithelial cells
 28. A method of claim 25,wherein said cells are dried in droplets of drying buffer.
 29. A methodfor increasing survival of mammalian nucleated cells following dryingand rehydration, comprising: (a) introducing a heat shock protein into,or inducing production of a heat shock protein in, said cells, toproduce heat shock protein-loaded cells, (b) drying said heat shockprotein-loaded cells; and (c) rehydrating said cells, thereby increasingsurvival of the cells.
 30. A method of claim 29, wherein said heat shockprotein is p26 from Artemia franciscana.
 31. A method of claim 29,wherein said heat shock protein is introduced into said cells byincubating said cells in a medium comprising said heat shock protein.32. A method of claim 29, wherein said heat shock protein is induced insaid cells by raising the temperature of medium contacting the cells to42-44° C. for one hour, and then allowing the temperature of the mediumto lower to 36-38° C.
 33. A method of claim 29, wherein said heat shockprotein is introduced into said cells by introducing into said cells anucleic acid sequence comprising a promoter operably linked to asequence encoding said heat shock protein.
 34. A method of claim 29,wherein said cells are selected from the group consisting of stem cells,immune system cells, and epithelial cells.
 35. A method of claim 29,wherein said cells are dried in droplets of drying buffer.
 36. A methodfor increasing survival of mammalian nucleated cells following dryingand rehydration, provided said cells are not 293 cells or B cells,comprising: (a) incubating said cells with a compound selected fromarbutin and hydroquinone, to produce arbutin- or hydroquinone-loadedcells, (b) drying said arbutin- or hydroquinone-loaded cells, and (c)rehydrating said cells, thereby increasing survival of the cells.
 37. Amethod of claim 36, wherein said compound of step (a) is arbutin.
 38. Anisolated mammalian nucleated cell comprising a disaccharide and acompound selected from the group consisting of arbutin and hydroquinone.39. An isolated mammalian nucleated cell of claim 38, wherein saidcompound is arbutin.
 40. A mammalian nucleated cell of claim 38, whereinsaid cell is dried.
 41. A mammalian nucleated cell of claim 38, furthercomprising an apoptosis inhibitor.
 42. A mammalian nucleated cell ofclaim 38, further comprising a heat shock protein.
 43. A mammaliannucleated cell of claim 38, wherein said disaccharide is trehalose. 44.An isolated dried mammalian nucleated cell comprising a disaccharide andan exogenous heat shock protein.
 45. A dried mammalian nucleated cell ofclaim 44, wherein said disaccharide is trehalose.
 46. A isolated, driedmammalian nucleated cell comprising a disaccharide and an exogenousapoptosis inhibitor.
 47. A dried mammalian nucleated cell of claim 46,wherein said disaccharide is trehalose.